From df73e1cc8142afda7d0d0d5da9a765fcd43c0f2d Mon Sep 17 00:00:00 2001
From: Jean Mainguy <jean.mainguy@inra.fr>
Date: Mon, 17 Oct 2022 14:57:10 +0200
Subject: [PATCH 001/194] Add prodigal module #163
---
modules/prodigal.nf | 18 ++++++++++++++++++
1 file changed, 18 insertions(+)
create mode 100644 modules/prodigal.nf
diff --git a/modules/prodigal.nf b/modules/prodigal.nf
new file mode 100644
index 0000000..1a9946c
--- /dev/null
+++ b/modules/prodigal.nf
@@ -0,0 +1,18 @@
+process PRODIGAL {
+ tag "${meta.id}"
+
+ input:
+ tuple val(meta), file(assembly_file)
+
+ output:
+ tuple val(meta), path("PROKKA_${meta.id}"), emit: prokka_results
+ path "v_prodigal.txt", emit: v_prokka
+
+ script:
+ """
+ prodigal -i ${assembly_file} -c -p meta -f gff -a prodigal.faa -o prodigal.gff
+
+ prodigal -v 2> v_prodigal.txt
+
+ """
+}
\ No newline at end of file
--
GitLab
From 478dd8fe1f205a52de9c8890daad843dc4aacebc Mon Sep 17 00:00:00 2001
From: Jean Mainguy <jean.mainguy@inra.fr>
Date: Mon, 17 Oct 2022 15:51:34 +0200
Subject: [PATCH 002/194] Add barrnap module see issue #163
---
modules/barnap.nf | 18 ++++++++++++++++++
1 file changed, 18 insertions(+)
create mode 100644 modules/barnap.nf
diff --git a/modules/barnap.nf b/modules/barnap.nf
new file mode 100644
index 0000000..0b88b76
--- /dev/null
+++ b/modules/barnap.nf
@@ -0,0 +1,18 @@
+process PRODIGAL {
+ tag "${meta.id}"
+
+ input:
+ tuple val(meta), file(assembly_file)
+
+ output:
+ tuple val(meta), path("barrnap.gff"), emit: barrnap_gff
+ path "v_barrnap.txt", emit: v_barrnap
+
+ script:
+ """
+ barrnap -o barrnap.gff --threads ${task.cpus} ${assembly_file}
+
+ barrnap --version 2> v_barrnap.txt
+
+ """
+}
--
GitLab
From efc393d920dc0506cf4e96d816473c6d50648d6d Mon Sep 17 00:00:00 2001
From: Jean Mainguy <jean.mainguy@inra.fr>
Date: Mon, 17 Oct 2022 15:52:13 +0200
Subject: [PATCH 003/194] Fix typo in prodigal module see issue #163
---
modules/prodigal.nf | 5 +++--
1 file changed, 3 insertions(+), 2 deletions(-)
diff --git a/modules/prodigal.nf b/modules/prodigal.nf
index 1a9946c..be69d07 100644
--- a/modules/prodigal.nf
+++ b/modules/prodigal.nf
@@ -5,8 +5,9 @@ process PRODIGAL {
tuple val(meta), file(assembly_file)
output:
- tuple val(meta), path("PROKKA_${meta.id}"), emit: prokka_results
- path "v_prodigal.txt", emit: v_prokka
+ tuple val(meta), path("prodigal.gff"), emit: prodigal_gff
+ tuple val(meta), path("prodigal.faa"), emit: prodigal_faa
+ path "v_prodigal.txt", emit: v_prodigal
script:
"""
--
GitLab
From 72577b733a62a866ee1f93ba5d46d5afd9492dea Mon Sep 17 00:00:00 2001
From: Jean Mainguy <jean.mainguy@inra.fr>
Date: Mon, 17 Oct 2022 15:55:34 +0200
Subject: [PATCH 004/194] fix module name
---
modules/barnap.nf | 2 +-
1 file changed, 1 insertion(+), 1 deletion(-)
diff --git a/modules/barnap.nf b/modules/barnap.nf
index 0b88b76..302ab6d 100644
--- a/modules/barnap.nf
+++ b/modules/barnap.nf
@@ -1,4 +1,4 @@
-process PRODIGAL {
+process BARRNAP {
tag "${meta.id}"
input:
--
GitLab
From ac29aef2c33a515efb617199aa26c05e5486527a Mon Sep 17 00:00:00 2001
From: Jean Mainguy <jean.mainguy@inra.fr>
Date: Mon, 17 Oct 2022 16:18:15 +0200
Subject: [PATCH 005/194] fix typo in barrnap name module
---
modules/{barnap.nf => barrnap.nf} | 0
1 file changed, 0 insertions(+), 0 deletions(-)
rename modules/{barnap.nf => barrnap.nf} (100%)
diff --git a/modules/barnap.nf b/modules/barrnap.nf
similarity index 100%
rename from modules/barnap.nf
rename to modules/barrnap.nf
--
GitLab
From 780d2d386d9886108064a8ddafad577e8058177c Mon Sep 17 00:00:00 2001
From: Jean Mainguy <jean.mainguy@inra.fr>
Date: Mon, 17 Oct 2022 17:10:09 +0200
Subject: [PATCH 006/194] structuring step 04 with prodigal + barrnap approach
---
main.nf | 17 +++++++++--------
subworkflows/04_structural_annot.nf | 26 ++++++++++++++++----------
2 files changed, 25 insertions(+), 18 deletions(-)
diff --git a/main.nf b/main.nf
index 2a20f0b..788b67f 100644
--- a/main.nf
+++ b/main.nf
@@ -446,17 +446,18 @@ workflow {
if ( !params.stop_at_clean && !params.stop_at_assembly && !params.stop_at_filtering ) {
S04_STRUCTURAL_ANNOT ( ch_assembly )
- ch_prokka_ffn = S04_STRUCTURAL_ANNOT.out.ffn
- ch_prokka_faa = S04_STRUCTURAL_ANNOT.out.faa
- ch_prokka_gff = S04_STRUCTURAL_ANNOT.out.gff
- ch_prokka_fna = S04_STRUCTURAL_ANNOT.out.fna
- ch_prokka_report = S04_STRUCTURAL_ANNOT.out.report
+ // ch_prokka_ffn = S04_STRUCTURAL_ANNOT.out.ffn
+ // ch_prokka_faa = S04_STRUCTURAL_ANNOT.out.faa
+ // ch_prokka_gff = S04_STRUCTURAL_ANNOT.out.gff
+ // ch_prokka_fna = S04_STRUCTURAL_ANNOT.out.fna
+ // ch_prokka_report = S04_STRUCTURAL_ANNOT.out.report
- ch_contigs_and_reads = ch_prokka_fna
- .join(ch_reads, remainder: true)
- ch_prot_length = S04_STRUCTURAL_ANNOT.out.prot_length
+ // ch_contigs_and_reads = ch_prokka_fna
+ // .join(ch_reads, remainder: true)
+ // ch_prot_length = S04_STRUCTURAL_ANNOT.out.prot_length
ch_software_versions_S04 = S04_STRUCTURAL_ANNOT.out.software_versions
+ // exit 0, 'Exit now... S04_STRUCTURAL_ANNOT being constructed '
}
ch_bam = Channel.empty()
diff --git a/subworkflows/04_structural_annot.nf b/subworkflows/04_structural_annot.nf
index 61bff36..ba3d4fe 100644
--- a/subworkflows/04_structural_annot.nf
+++ b/subworkflows/04_structural_annot.nf
@@ -1,19 +1,25 @@
-include { PROKKA; RENAME_CONTIGS_AND_GENES } from '../modules/prokka'
+include { PRODIGAL } from '../modules/prodigal'
+include { BARRNAP } from '../modules/barrnap'
workflow STEP_04_STRUCTURAL_ANNOT {
take: assembly
main:
- PROKKA( assembly )
- ch_software_versions = PROKKA.out.v_prokka.first()
- RENAME_CONTIGS_AND_GENES(PROKKA.out.prokka_results)
+ PRODIGAL( assembly )
+ BARRNAP( assembly )
+ ch_software_versions = PRODIGAL.out.v_prodigal.first()
+
+
+ ch_software_versions = PRODIGAL.out.v_prodigal.first().mix( BARRNAP.out.v_barrnap.first())
+
+ // RENAME_CONTIGS_AND_GENES(PROKKA.out.prokka_results)
emit:
- report = PROKKA.out.report
- fna = RENAME_CONTIGS_AND_GENES.out.fna
- ffn = RENAME_CONTIGS_AND_GENES.out.ffn
- gff = RENAME_CONTIGS_AND_GENES.out.gff
- faa = RENAME_CONTIGS_AND_GENES.out.faa
- prot_length = RENAME_CONTIGS_AND_GENES.out.prot_length
+ // report = PROKKA.out.report
+ // fna = RENAME_CONTIGS_AND_GENES.out.fna
+ // ffn = RENAME_CONTIGS_AND_GENES.out.ffn
+ // gff = RENAME_CONTIGS_AND_GENES.out.gff
+ // faa = RENAME_CONTIGS_AND_GENES.out.faa
+ // prot_length = RENAME_CONTIGS_AND_GENES.out.prot_length
software_versions = ch_software_versions
}
\ No newline at end of file
--
GitLab
From 898379888ec9c26f53afdd86c192897fb33b7ed0 Mon Sep 17 00:00:00 2001
From: Jean Mainguy <jean.mainguy@inra.fr>
Date: Mon, 17 Oct 2022 17:10:43 +0200
Subject: [PATCH 007/194] fix barnnap output
---
modules/barrnap.nf | 2 +-
1 file changed, 1 insertion(+), 1 deletion(-)
diff --git a/modules/barrnap.nf b/modules/barrnap.nf
index 302ab6d..44cbd5e 100644
--- a/modules/barrnap.nf
+++ b/modules/barrnap.nf
@@ -10,7 +10,7 @@ process BARRNAP {
script:
"""
- barrnap -o barrnap.gff --threads ${task.cpus} ${assembly_file}
+ barrnap --threads ${task.cpus} ${assembly_file} > barrnap.gff
barrnap --version 2> v_barrnap.txt
--
GitLab
From d2e2344c12be0a659633daf69da7daf5ce874463 Mon Sep 17 00:00:00 2001
From: Jean Mainguy <jean.mainguy@inra.fr>
Date: Wed, 19 Oct 2022 10:35:40 +0200
Subject: [PATCH 008/194] Add trnascan and pyfastx in conda recipe
issue #163
---
env/metagWGS.yml | 4 +++-
1 file changed, 3 insertions(+), 1 deletion(-)
diff --git a/env/metagWGS.yml b/env/metagWGS.yml
index e5a3375..af1e578 100644
--- a/env/metagWGS.yml
+++ b/env/metagWGS.yml
@@ -27,4 +27,6 @@ dependencies:
- subread=2.0.1
- flye=2.9.1
- hifiasm_meta=hamtv0.3
- - plotly
\ No newline at end of file
+ - plotly
+ - trnascan-se=2.0.11
+ - pyfastx
\ No newline at end of file
--
GitLab
From 4e805a4e95230e8e27d35fe322cc415a433b02ca Mon Sep 17 00:00:00 2001
From: Jean Mainguy <jean.mainguy@inra.fr>
Date: Wed, 19 Oct 2022 11:45:15 +0200
Subject: [PATCH 009/194] Add trnascan_se module issue #163
---
modules/trnascan_se.nf | 19 +++++++++++++++++++
subworkflows/04_structural_annot.nf | 6 +++++-
2 files changed, 24 insertions(+), 1 deletion(-)
create mode 100644 modules/trnascan_se.nf
diff --git a/modules/trnascan_se.nf b/modules/trnascan_se.nf
new file mode 100644
index 0000000..e292e3e
--- /dev/null
+++ b/modules/trnascan_se.nf
@@ -0,0 +1,19 @@
+process TRNASCAN_SE {
+ tag "${meta.id}"
+
+ input:
+ tuple val(meta), file(assembly_file)
+
+ output:
+ tuple val(meta), path("trnascan_se.gff"), emit: trnascan_gff
+ path "v_tRNAscan.txt", emit: v_tRNAscan
+
+ script:
+ """
+ tRNAscan-SE -B --gff trnascan_se.gff --thread ${task.cpus} --stats trnascan_se.log ${assembly_file}
+
+ tRNAscan-SE -h 2> v_tRNAscan.txt
+
+
+ """
+}
diff --git a/subworkflows/04_structural_annot.nf b/subworkflows/04_structural_annot.nf
index ba3d4fe..4e6687b 100644
--- a/subworkflows/04_structural_annot.nf
+++ b/subworkflows/04_structural_annot.nf
@@ -1,5 +1,6 @@
include { PRODIGAL } from '../modules/prodigal'
include { BARRNAP } from '../modules/barrnap'
+include { TRNASCAN_SE } from '../modules/trnascan_se'
workflow STEP_04_STRUCTURAL_ANNOT {
take: assembly
@@ -7,10 +8,13 @@ workflow STEP_04_STRUCTURAL_ANNOT {
main:
PRODIGAL( assembly )
BARRNAP( assembly )
+ TRNASCAN_SE( assembly )
+
ch_software_versions = PRODIGAL.out.v_prodigal.first()
- ch_software_versions = PRODIGAL.out.v_prodigal.first().mix( BARRNAP.out.v_barrnap.first())
+ ch_software_versions = PRODIGAL.out.v_prodigal.first().mix( BARRNAP.out.v_barrnap.first(),
+ TRNASCAN_SE.out.v_tRNAscan.first())
// RENAME_CONTIGS_AND_GENES(PROKKA.out.prokka_results)
--
GitLab
From 8d92905cfc45da05590d9e4eb75d4f0ed14761bb Mon Sep 17 00:00:00 2001
From: Jean Mainguy <jean.mainguy@inra.fr>
Date: Wed, 19 Oct 2022 17:48:21 +0200
Subject: [PATCH 010/194] simplify channel name
---
modules/barrnap.nf | 2 +-
modules/prodigal.nf | 4 ++--
modules/trnascan_se.nf | 2 +-
3 files changed, 4 insertions(+), 4 deletions(-)
diff --git a/modules/barrnap.nf b/modules/barrnap.nf
index 44cbd5e..16013a0 100644
--- a/modules/barrnap.nf
+++ b/modules/barrnap.nf
@@ -5,7 +5,7 @@ process BARRNAP {
tuple val(meta), file(assembly_file)
output:
- tuple val(meta), path("barrnap.gff"), emit: barrnap_gff
+ tuple val(meta), path("barrnap.gff"), emit: gff
path "v_barrnap.txt", emit: v_barrnap
script:
diff --git a/modules/prodigal.nf b/modules/prodigal.nf
index be69d07..63bde69 100644
--- a/modules/prodigal.nf
+++ b/modules/prodigal.nf
@@ -5,8 +5,8 @@ process PRODIGAL {
tuple val(meta), file(assembly_file)
output:
- tuple val(meta), path("prodigal.gff"), emit: prodigal_gff
- tuple val(meta), path("prodigal.faa"), emit: prodigal_faa
+ tuple val(meta), path("prodigal.gff"), emit: gff
+ tuple val(meta), path("prodigal.faa"), emit: faa
path "v_prodigal.txt", emit: v_prodigal
script:
diff --git a/modules/trnascan_se.nf b/modules/trnascan_se.nf
index e292e3e..b690c7c 100644
--- a/modules/trnascan_se.nf
+++ b/modules/trnascan_se.nf
@@ -5,7 +5,7 @@ process TRNASCAN_SE {
tuple val(meta), file(assembly_file)
output:
- tuple val(meta), path("trnascan_se.gff"), emit: trnascan_gff
+ tuple val(meta), path("trnascan_se.gff"), emit: gff
path "v_tRNAscan.txt", emit: v_tRNAscan
script:
--
GitLab
From daf624e354e19c03aa5d6360717d53510d3b5307 Mon Sep 17 00:00:00 2001
From: Jean Mainguy <jean.mainguy@inra.fr>
Date: Wed, 19 Oct 2022 18:03:14 +0200
Subject: [PATCH 011/194] Add process and script to merge gff annotations issue
#163
---
bin/merge_annotations.py | 178 ++++++++++++++++++++++++++++
subworkflows/04_structural_annot.nf | 12 ++
2 files changed, 190 insertions(+)
create mode 100755 bin/merge_annotations.py
diff --git a/bin/merge_annotations.py b/bin/merge_annotations.py
new file mode 100755
index 0000000..8d3b4fa
--- /dev/null
+++ b/bin/merge_annotations.py
@@ -0,0 +1,178 @@
+#!/usr/bin/env python3
+
+"""
+Combine structural annotations made from different gff.
+
+In case of collapsing annotations RNA annotation are prefered to CDS annotations.
+
+:Example:
+python template.py -v
+"""
+
+# Metadata
+__author__ = 'Mainguy Jean - Plateforme bioinformatique Toulouse'
+__copyright__ = 'Copyright (C) 2020 INRAE'
+__license__ = 'GNU General Public License'
+__version__ = '0.1'
+__email__ = 'support.bioinfo.genotoul@inra.fr'
+__status__ = 'dev'
+
+
+from argparse import ArgumentParser, ArgumentDefaultsHelpFormatter, FileType
+import logging
+import gzip
+import csv
+from collections import defaultdict
+
+def read_file_and_ignore_hashtag(file_path):
+ proper_open = gzip.open if file_path.endswith('.gz') else open
+ with proper_open(file_path, 'rt') as fl:
+ for line in fl:
+ if line.startswith('#'):
+ continue
+ yield line
+
+def group_annotations_by_contigs(*gff_annotations):
+
+ contig2annotations = defaultdict(list)
+ for annotations in gff_annotations:
+ for annotation in annotations:
+ contig2annotations[annotation['seqname']].append(annotation)
+
+ return contig2annotations
+
+
+def parse_gff_file(gff_file, feature_to_keep=False):
+
+ gff_headers = ("seqname", "_3", "feature", "start", "end", "_2", "strand", "_1", "attribute")
+
+ gff_annotations = csv.DictReader(read_file_and_ignore_hashtag(gff_file),
+ delimiter='\t',
+ fieldnames=gff_headers)
+
+ if feature_to_keep:
+ logging.info(f'Keeping only {feature_to_keep} feature from {gff_file}')
+ gff_annotations = (annotation for annotation in gff_annotations if annotation['feature'] == feature_to_keep)
+
+ return gff_annotations
+
+def parse_arguments():
+ """Parse script arguments."""
+ parser = ArgumentParser(description="...",
+ formatter_class=ArgumentDefaultsHelpFormatter)
+
+ parser.add_argument('-c', '--cds', help='gff file with CDS annotations.', required=True)
+
+ parser.add_argument('-t', '--trna', help='gff file with tRNA annotations.', required=True)
+
+ parser.add_argument('-r', '--rrna', help='gff file with rRNA annotations.', required=True)
+
+ parser.add_argument('-o', '--gff_output', help='final gff file with merged annotations.', required=True)
+
+
+ parser.add_argument("-v", "--verbose", help="increase output verbosity",
+ action="store_true")
+
+ args = parser.parse_args()
+ return args
+
+def remove_overlapping_cds(cds_file, contig2rnas):
+
+ contig_annotations = []
+ current_contig = None
+
+ for cds in parse_gff_file(cds_file, 'CDS'):
+
+ reading_next_contig = cds['seqname'] != current_contig
+
+ if contig_annotations and reading_next_contig:
+
+ yield current_contig, contig_annotations
+ contig_annotations = []
+
+ current_contig = cds['seqname']
+
+ is_overlapping = False
+ for rna in contig2rnas[current_contig]:
+ if (int(rna['end']) < int(cds['start']) or int(rna['start']) > int(cds['end'])):
+ continue
+
+ else: # overlap -> remove cds
+ is_overlapping = True
+ overlap = f"[{max(rna['start'], cds['start'])},{min(rna['end'], cds['end'])}]"
+ rna_info = f"{rna['feature']} [{rna['start']}, {rna['end']}]"
+ cds_info = f"CDS [{cds['start']}, {cds['end']}]"
+ logging.info(f"overlap: {cds_info} overlapping with {rna_info} at {overlap} on contig={current_contig}")
+
+ if not is_overlapping:
+ contig_annotations.append(cds)
+
+ yield current_contig, contig_annotations
+
+def merging_cds_and_rna(cds_per_contig, contig2rnas):
+ contig_processed = []
+ for contig, cds_features in cds_per_contig:
+ contig_processed.append(contig)
+ rna_features = contig2rnas[contig]
+ yield contig, rna_features + cds_features
+
+ # check that all rrna contigs have been processed if not processe them
+ for contig, rnas in contig2rnas.items():
+ if contig not in contig_processed:
+ logging.info(f'{contig} has only rna annotation')
+ yield contig, rnas
+
+def writing_features_to_gff(annotations_per_contig, out_gff):
+
+ with open(out_gff, 'w') as fl:
+
+ fl.write("##gff-version 3\n")
+ for contig, features in annotations_per_contig:
+ logging.info(f"writing {contig} annotations to gff file")
+ fl.write(f"##sequence-region {contig}\n")
+
+ for feature in sorted(features, key=lambda x: int(x['start'])):
+ fl.write('\t'.join(feature.values())+"\n")
+
+def main():
+
+ args = parse_arguments()
+
+ if args.verbose:
+ logging.basicConfig(format="%(levelname)s: %(message)s", level=logging.DEBUG)
+ logging.info('Mode verbose ON')
+
+ else:
+ logging.basicConfig(format="%(levelname)s: %(message)s")
+
+
+ trna_file = args.trna
+ rrna_file = args.rrna
+ cds_file = args.cds
+
+ output_gff = args.gff_output
+
+ logging.info('Parsing rRNA annoations.')
+ rrna_annotations = parse_gff_file(rrna_file, 'rRNA')
+
+ logging.info('Parsing tRNA annoations.')
+ trna_annotations = parse_gff_file(trna_file, 'tRNA')
+
+ logging.info('Grouping RNA annoations by contig.')
+ contig2rnas = group_annotations_by_contigs(trna_annotations, rrna_annotations)
+
+ logging.info('Removing CDS annoations overlapping a RNA annotations.')
+ unoverlapping_cds_per_contig = remove_overlapping_cds(cds_file, contig2rnas)
+
+ logging.info('Merge CDS and RNA annotations by contig.')
+ annotation_per_contigs = merging_cds_and_rna(unoverlapping_cds_per_contig, contig2rnas)
+
+ logging.info(f'Writting CDS and RNA annotations to gff file {output_gff}.')
+ writing_features_to_gff(annotation_per_contigs, output_gff)
+
+
+
+
+
+if __name__ == '__main__':
+ main()
\ No newline at end of file
diff --git a/subworkflows/04_structural_annot.nf b/subworkflows/04_structural_annot.nf
index 4e6687b..a70b215 100644
--- a/subworkflows/04_structural_annot.nf
+++ b/subworkflows/04_structural_annot.nf
@@ -1,6 +1,8 @@
include { PRODIGAL } from '../modules/prodigal'
include { BARRNAP } from '../modules/barrnap'
include { TRNASCAN_SE } from '../modules/trnascan_se'
+include { MERGE_ANNOTATIONS } from '../modules/merge_annotations'
+
workflow STEP_04_STRUCTURAL_ANNOT {
take: assembly
@@ -10,6 +12,16 @@ workflow STEP_04_STRUCTURAL_ANNOT {
BARRNAP( assembly )
TRNASCAN_SE( assembly )
+
+ annotations_ch = PRODIGAL.out.gff.join(BARRNAP.out.gff)
+ .join(TRNASCAN_SE.out.gff)
+ annotations_ch.view()
+
+
+
+ MERGE_ANNOTATIONS(annotations_ch)
+
+
ch_software_versions = PRODIGAL.out.v_prodigal.first()
--
GitLab
From 0e927a96bdbbd3d3b84cf07741b7e38aebfcd997 Mon Sep 17 00:00:00 2001
From: Jean Mainguy <jean.mainguy@inra.fr>
Date: Wed, 19 Oct 2022 19:01:45 +0200
Subject: [PATCH 012/194] add ffn output on top of gff output
---
bin/merge_annotations.py | 51 +++++++++++++++++++++++++----
subworkflows/04_structural_annot.nf | 2 +-
2 files changed, 45 insertions(+), 8 deletions(-)
diff --git a/bin/merge_annotations.py b/bin/merge_annotations.py
index 8d3b4fa..91ebdb0 100755
--- a/bin/merge_annotations.py
+++ b/bin/merge_annotations.py
@@ -23,6 +23,8 @@ import logging
import gzip
import csv
from collections import defaultdict
+import pyfastx
+
def read_file_and_ignore_hashtag(file_path):
proper_open = gzip.open if file_path.endswith('.gz') else open
@@ -67,7 +69,11 @@ def parse_arguments():
parser.add_argument('-r', '--rrna', help='gff file with rRNA annotations.', required=True)
- parser.add_argument('-o', '--gff_output', help='final gff file with merged annotations.', required=True)
+ parser.add_argument('--gff_output', help='final gff file with all annotations.', default="all_annotation.gff")
+ parser.add_argument('--ffn_output', help='final ffn file with all annotations.', default="all_annotation.ffn")
+
+ parser.add_argument('--contig_seq', help='fasta file of contigs. Needed to extract gene annotations', required=True)
+
parser.add_argument("-v", "--verbose", help="increase output verbosity",
@@ -122,17 +128,44 @@ def merging_cds_and_rna(cds_per_contig, contig2rnas):
logging.info(f'{contig} has only rna annotation')
yield contig, rnas
-def writing_features_to_gff(annotations_per_contig, out_gff):
+def add_new_ID_tag(gff_attributes, new_id):
+ gff_attributes_no_id = ';'.join([attr for attr in gff_attributes.split(';') if attr.split('=')[0] != 'ID'])
+ return f"ID={new_id};{gff_attributes_no_id}"
+
+def get_Name_tag(gff_attributes):
+ for attr in gff_attributes.split(';'):
+ if attr.split('=')[0] == "Name":
+ return attr.split('=')[1]
+ return ''
+
+
+def writing_features_to_gff_and_ffn(annotations_per_contig, out_gff, fna_file, out_ffn):
- with open(out_gff, 'w') as fl:
+ contig_fa = pyfastx.Fasta(fna_file)
+
+
+ with open(out_gff, 'w') as fh_gff, open(out_ffn, 'w') as fh_ffn:
- fl.write("##gff-version 3\n")
+ fh_gff.write("##gff-version 3\n")
for contig, features in annotations_per_contig:
+ gene_count = defaultdict(int)
logging.info(f"writing {contig} annotations to gff file")
- fl.write(f"##sequence-region {contig}\n")
+ fh_gff.write(f"##sequence-region {contig}\n")
for feature in sorted(features, key=lambda x: int(x['start'])):
- fl.write('\t'.join(feature.values())+"\n")
+ gene_count[feature['feature']] += 1
+ new_id = f"{feature['seqname']}.{feature['feature']}_{gene_count[feature['feature']]}"
+
+ feature['attribute'] = add_new_ID_tag(feature['attribute'], new_id)
+
+ tag_name = get_Name_tag(feature['attribute'])
+ fh_gff.write('\t'.join(feature.values())+"\n")
+
+ feature_seq = contig_fa.fetch(contig, [int(feature['start']), int(feature['end'])])
+ fh_ffn.write(f">{new_id} {tag_name}\n")
+ fh_ffn.write(f"{feature_seq}\n")
+
+
def main():
@@ -152,6 +185,9 @@ def main():
output_gff = args.gff_output
+ fna_file = args.contig_seq
+ out_ffn = args.ffn_output
+
logging.info('Parsing rRNA annoations.')
rrna_annotations = parse_gff_file(rrna_file, 'rRNA')
@@ -168,11 +204,12 @@ def main():
annotation_per_contigs = merging_cds_and_rna(unoverlapping_cds_per_contig, contig2rnas)
logging.info(f'Writting CDS and RNA annotations to gff file {output_gff}.')
- writing_features_to_gff(annotation_per_contigs, output_gff)
+ writing_features_to_gff_and_ffn(annotation_per_contigs, output_gff, fna_file, out_ffn)
+
if __name__ == '__main__':
main()
\ No newline at end of file
diff --git a/subworkflows/04_structural_annot.nf b/subworkflows/04_structural_annot.nf
index a70b215..e420d55 100644
--- a/subworkflows/04_structural_annot.nf
+++ b/subworkflows/04_structural_annot.nf
@@ -13,7 +13,7 @@ workflow STEP_04_STRUCTURAL_ANNOT {
TRNASCAN_SE( assembly )
- annotations_ch = PRODIGAL.out.gff.join(BARRNAP.out.gff)
+ annotations_ch = assembly.join(PRODIGAL.out.gff).join(BARRNAP.out.gff)
.join(TRNASCAN_SE.out.gff)
annotations_ch.view()
--
GitLab
From e5dae7acc2b266df8092dd68448d90e16c95572a Mon Sep 17 00:00:00 2001
From: Jean Mainguy <jean.mainguy@inra.fr>
Date: Thu, 20 Oct 2022 09:31:10 +0200
Subject: [PATCH 013/194] process to launch python script to merge gff files
---
modules/merge_annotations.nf | 15 +++++++++++++++
1 file changed, 15 insertions(+)
create mode 100644 modules/merge_annotations.nf
diff --git a/modules/merge_annotations.nf b/modules/merge_annotations.nf
new file mode 100644
index 0000000..4cbbd82
--- /dev/null
+++ b/modules/merge_annotations.nf
@@ -0,0 +1,15 @@
+process MERGE_ANNOTATIONS {
+ tag "${meta.id}"
+
+ input:
+ tuple val(meta), file(assembly_file), file(cds_gff), file(rrna_gff), file(trna_gff)
+
+ output:
+
+
+ script:
+ """
+ merge_annotations.py -c $cds_gff -r $rrna_gff -t $trna_gff -v --contig_seq $assembly_file \
+ --ffn_output ${meta.sample}.ffn --gff_output ${meta.sample}.gff
+ """
+}
\ No newline at end of file
--
GitLab
From dfd1ce639f6fa50343b7406cc8c115e821d46907 Mon Sep 17 00:00:00 2001
From: Jean Mainguy <jean.mainguy@inra.fr>
Date: Thu, 20 Oct 2022 13:37:32 +0200
Subject: [PATCH 014/194] fix strand and zero base position when extracting seq
---
bin/merge_annotations.py | 112 +++++++++++++++++++++++++--------------
1 file changed, 73 insertions(+), 39 deletions(-)
diff --git a/bin/merge_annotations.py b/bin/merge_annotations.py
index 91ebdb0..5434cdb 100755
--- a/bin/merge_annotations.py
+++ b/bin/merge_annotations.py
@@ -24,9 +24,36 @@ import gzip
import csv
from collections import defaultdict
import pyfastx
+from Bio.Seq import Seq
+def parse_arguments():
+ """Parse script arguments."""
+ parser = ArgumentParser(description="...",
+ formatter_class=ArgumentDefaultsHelpFormatter)
+
+ parser.add_argument('-c', '--cds', help='gff file with CDS annotations.', required=True)
+
+ parser.add_argument('-t', '--trna', help='gff file with tRNA annotations.', required=True)
+
+ parser.add_argument('-r', '--rrna', help='gff file with rRNA annotations.', required=True)
+
+ parser.add_argument('--gff_output', help='final gff file with all annotations.', default="all_annotation.gff")
+
+ parser.add_argument('--ffn_output', help='final ffn file with all annotations.', default="all_annotation.ffn")
+
+ parser.add_argument('--faa_output', help='final faa file with all CDS annotations in amino acid.', default="all_annotation.faa")
+
+ parser.add_argument('--contig_seq', help='fasta file of contigs. Needed to extract gene annotations', required=True)
+
+ parser.add_argument("-v", "--verbose", help="increase output verbosity",
+ action="store_true")
+
+ args = parser.parse_args()
+ return args
+
def read_file_and_ignore_hashtag(file_path):
+
proper_open = gzip.open if file_path.endswith('.gz') else open
with proper_open(file_path, 'rt') as fl:
for line in fl:
@@ -58,30 +85,6 @@ def parse_gff_file(gff_file, feature_to_keep=False):
return gff_annotations
-def parse_arguments():
- """Parse script arguments."""
- parser = ArgumentParser(description="...",
- formatter_class=ArgumentDefaultsHelpFormatter)
-
- parser.add_argument('-c', '--cds', help='gff file with CDS annotations.', required=True)
-
- parser.add_argument('-t', '--trna', help='gff file with tRNA annotations.', required=True)
-
- parser.add_argument('-r', '--rrna', help='gff file with rRNA annotations.', required=True)
-
- parser.add_argument('--gff_output', help='final gff file with all annotations.', default="all_annotation.gff")
- parser.add_argument('--ffn_output', help='final ffn file with all annotations.', default="all_annotation.ffn")
-
- parser.add_argument('--contig_seq', help='fasta file of contigs. Needed to extract gene annotations', required=True)
-
-
-
- parser.add_argument("-v", "--verbose", help="increase output verbosity",
- action="store_true")
-
- args = parser.parse_args()
- return args
-
def remove_overlapping_cds(cds_file, contig2rnas):
contig_annotations = []
@@ -116,6 +119,7 @@ def remove_overlapping_cds(cds_file, contig2rnas):
yield current_contig, contig_annotations
def merging_cds_and_rna(cds_per_contig, contig2rnas):
+
contig_processed = []
for contig, cds_features in cds_per_contig:
contig_processed.append(contig)
@@ -129,21 +133,22 @@ def merging_cds_and_rna(cds_per_contig, contig2rnas):
yield contig, rnas
def add_new_ID_tag(gff_attributes, new_id):
+
gff_attributes_no_id = ';'.join([attr for attr in gff_attributes.split(';') if attr.split('=')[0] != 'ID'])
return f"ID={new_id};{gff_attributes_no_id}"
-def get_Name_tag(gff_attributes):
- for attr in gff_attributes.split(';'):
- if attr.split('=')[0] == "Name":
+
+def get_tag_value(gff_attribute, tag):
+
+ for attr in gff_attribute.split(';'):
+ if attr.split('=')[0] == tag:
return attr.split('=')[1]
return ''
-
def writing_features_to_gff_and_ffn(annotations_per_contig, out_gff, fna_file, out_ffn):
contig_fa = pyfastx.Fasta(fna_file)
-
with open(out_gff, 'w') as fh_gff, open(out_ffn, 'w') as fh_ffn:
fh_gff.write("##gff-version 3\n")
@@ -158,13 +163,41 @@ def writing_features_to_gff_and_ffn(annotations_per_contig, out_gff, fna_file, o
feature['attribute'] = add_new_ID_tag(feature['attribute'], new_id)
- tag_name = get_Name_tag(feature['attribute'])
+ tag_name = get_tag_value(feature['attribute'], 'Name')
fh_gff.write('\t'.join(feature.values())+"\n")
- feature_seq = contig_fa.fetch(contig, [int(feature['start']), int(feature['end'])])
- fh_ffn.write(f">{new_id} {tag_name}\n")
- fh_ffn.write(f"{feature_seq}\n")
+ # feature_seq = contig_fa.fetch(contig, [int(feature['start']), int(feature['end'])])
+ #
+
+ start_zero, end_zero = int(feature['start'])-1, int(feature['end'])
+
+ if feature['strand'] == "+":
+ feature_seq = contig_fa[contig][start_zero:end_zero].seq
+ else:
+ # reverse complement the sequence
+ feature_seq = contig_fa[contig][start_zero:end_zero].antisense
+
+ fh_ffn.write(f">{new_id} {tag_name}\n{feature_seq}\n")
+
+
+ # if feature['feature'] == "CDS":
+
+ # aa_seq = Seq(feature_seq).translate(table=translation_table, cds=True)
+
+ # fh_faa.write(f'>{new_id}\n{aa_seq}\n')
+
+
+# def filter_faa_file(annotation_per_contigs, faa_file, out_faa):
+
+# protein_fa = pyfastx.Fasta(faa_file)
+# all_names = [set(protein_fa.keys().filter())]
+
+
+# for contig, features in annotation_per_contigs:
+
+
+# yield contig, features
def main():
@@ -187,6 +220,9 @@ def main():
fna_file = args.contig_seq
out_ffn = args.ffn_output
+ out_faa = args.faa_output
+
+ faa_file = "prodigal.faa"
logging.info('Parsing rRNA annoations.')
rrna_annotations = parse_gff_file(rrna_file, 'rRNA')
@@ -203,13 +239,11 @@ def main():
logging.info('Merge CDS and RNA annotations by contig.')
annotation_per_contigs = merging_cds_and_rna(unoverlapping_cds_per_contig, contig2rnas)
- logging.info(f'Writting CDS and RNA annotations to gff file {output_gff}.')
- writing_features_to_gff_and_ffn(annotation_per_contigs, output_gff, fna_file, out_ffn)
-
-
-
-
+ # annotation_per_contigs = filter_faa_file(annotation_per_contigs, faa_file, out_faa)
+ logging.info(f'Writting CDS and RNA annotations to gff file: {output_gff}.')
+ writing_features_to_gff_and_ffn(annotation_per_contigs, output_gff, fna_file, out_ffn) #, out_faa)
+
if __name__ == '__main__':
main()
\ No newline at end of file
--
GitLab
From b00645a569d5ebfe42b10581d220c2365738bbf4 Mon Sep 17 00:00:00 2001
From: Jean Mainguy <jean.mainguy@inra.fr>
Date: Thu, 20 Oct 2022 14:35:49 +0200
Subject: [PATCH 015/194] add faa file filtering from prodigal
---
bin/merge_annotations.py | 69 +++++++++++++++++++++++++---------------
1 file changed, 44 insertions(+), 25 deletions(-)
diff --git a/bin/merge_annotations.py b/bin/merge_annotations.py
index 5434cdb..e6a5c59 100755
--- a/bin/merge_annotations.py
+++ b/bin/merge_annotations.py
@@ -145,11 +145,13 @@ def get_tag_value(gff_attribute, tag):
return attr.split('=')[1]
return ''
-def writing_features_to_gff_and_ffn(annotations_per_contig, out_gff, fna_file, out_ffn):
+def writing_features_to_gff_ffn_faa(annotations_per_contig, out_gff, fna_file, out_ffn, faa_file, out_faa):
contig_fa = pyfastx.Fasta(fna_file)
+ protein_fa = pyfastx.Fasta(faa_file)
- with open(out_gff, 'w') as fh_gff, open(out_ffn, 'w') as fh_ffn:
+
+ with open(out_gff, 'w') as fh_gff, open(out_ffn, 'w') as fh_ffn, open(out_faa, 'w') as fh_faa:
fh_gff.write("##gff-version 3\n")
for contig, features in annotations_per_contig:
@@ -161,43 +163,60 @@ def writing_features_to_gff_and_ffn(annotations_per_contig, out_gff, fna_file, o
gene_count[feature['feature']] += 1
new_id = f"{feature['seqname']}.{feature['feature']}_{gene_count[feature['feature']]}"
+ start, end = int(feature['start']), int(feature['end'])
+
+ # write faa
+ if feature['feature'] == "CDS":
+ gff_id = get_tag_value(feature['attribute'], 'ID')
+
+ # ID in gff from prodigal is <contig_number>_<cds_number>
+ # seq name in faa from prodigal is <contig_name>_<cds_number>
+ cds_number = gff_id.split('_')[-1]
+ cds_prodigal_id = f"{contig}_{cds_number}"
+ fh_faa.write(f">{new_id} {start}-{end}\n{protein_fa[cds_prodigal_id]}\n")
+
+
+ # Write gff and ffn
feature['attribute'] = add_new_ID_tag(feature['attribute'], new_id)
tag_name = get_tag_value(feature['attribute'], 'Name')
fh_gff.write('\t'.join(feature.values())+"\n")
-
- # feature_seq = contig_fa.fetch(contig, [int(feature['start']), int(feature['end'])])
- #
-
-
- start_zero, end_zero = int(feature['start'])-1, int(feature['end'])
if feature['strand'] == "+":
- feature_seq = contig_fa[contig][start_zero:end_zero].seq
+ feature_seq = contig_fa[contig][start-1:end].seq
else:
- # reverse complement the sequence
- feature_seq = contig_fa[contig][start_zero:end_zero].antisense
+ # minus strand: reverse complement the sequence
+ feature_seq = contig_fa[contig][start-1:end].antisense
- fh_ffn.write(f">{new_id} {tag_name}\n{feature_seq}\n")
+ fh_ffn.write(f">{new_id} {start}-{end} {tag_name}\n{feature_seq}\n")
- # if feature['feature'] == "CDS":
-
- # aa_seq = Seq(feature_seq).translate(table=translation_table, cds=True)
-
- # fh_faa.write(f'>{new_id}\n{aa_seq}\n')
-
+
+def filter_faa_file(annotation_per_contigs, faa_file, out_faa):
-# def filter_faa_file(annotation_per_contigs, faa_file, out_faa):
+ protein_fa = pyfastx.Fasta(faa_file)
-# protein_fa = pyfastx.Fasta(faa_file)
-# all_names = [set(protein_fa.keys().filter())]
+ for contig, features in annotation_per_contigs:
+
+ for feature in features:
+ new_id = f"{feature['seqname']}.{feature['feature']}_{gene_count[feature['feature']]}"
+
+ gff_id = get_tag_value(feature['attribute'], 'ID')
+ # ID in gff from prodigal is <contig_number>_<cds_number>
+ # seq name from prodigal is <contig_name>_<cds_number>
+ cds_number = gff_id.split('_')[-1]
+ cds_prodigal_id = f"{contig}_{cds_number}"
+ print(cds_prodigal_id, new_id)
+ print(protein_fa[cds_prodigal_id])
+
+
+
+
+ input()
-
-# for contig, features in annotation_per_contigs:
-# yield contig, features
+ yield contig, features
def main():
@@ -242,7 +261,7 @@ def main():
# annotation_per_contigs = filter_faa_file(annotation_per_contigs, faa_file, out_faa)
logging.info(f'Writting CDS and RNA annotations to gff file: {output_gff}.')
- writing_features_to_gff_and_ffn(annotation_per_contigs, output_gff, fna_file, out_ffn) #, out_faa)
+ writing_features_to_gff_ffn_faa(annotation_per_contigs, output_gff, fna_file, out_ffn, faa_file, out_faa)
if __name__ == '__main__':
--
GitLab
From b7de39922bb5b1c9545a47509a340fd0a15ea79a Mon Sep 17 00:00:00 2001
From: Jean Mainguy <jean.mainguy@inra.fr>
Date: Thu, 20 Oct 2022 14:37:34 +0200
Subject: [PATCH 016/194] improve presentation string
---
bin/merge_annotations.py | 41 ++++++++--------------------------------
1 file changed, 8 insertions(+), 33 deletions(-)
diff --git a/bin/merge_annotations.py b/bin/merge_annotations.py
index e6a5c59..91af848 100755
--- a/bin/merge_annotations.py
+++ b/bin/merge_annotations.py
@@ -3,10 +3,11 @@
"""
Combine structural annotations made from different gff.
-In case of collapsing annotations RNA annotation are prefered to CDS annotations.
+In case of collapsing annotations, RNA annotation are prefered to CDS annotations.
+Faa file from prodigal is processed to filter out overlapping sequences.
:Example:
-python template.py -v
+merge_annotations.py -c prodigal.gff -r barrnap.gff -t trnascan_se.gff --contig_seq contigs.fasta --faa_file prodigal.faa
"""
# Metadata
@@ -37,14 +38,17 @@ def parse_arguments():
parser.add_argument('-t', '--trna', help='gff file with tRNA annotations.', required=True)
parser.add_argument('-r', '--rrna', help='gff file with rRNA annotations.', required=True)
+
+ parser.add_argument('--contig_seq', help='fasta file of contigs. Needed to extract gene sequence', required=True)
+
+ parser.add_argument('--faa_file', help='fasta file of protein sequences generated by prodigal.', required=True)
parser.add_argument('--gff_output', help='final gff file with all annotations.', default="all_annotation.gff")
parser.add_argument('--ffn_output', help='final ffn file with all annotations.', default="all_annotation.ffn")
parser.add_argument('--faa_output', help='final faa file with all CDS annotations in amino acid.', default="all_annotation.faa")
-
- parser.add_argument('--contig_seq', help='fasta file of contigs. Needed to extract gene annotations', required=True)
+
parser.add_argument("-v", "--verbose", help="increase output verbosity",
action="store_true")
@@ -191,33 +195,6 @@ def writing_features_to_gff_ffn_faa(annotations_per_contig, out_gff, fna_file, o
fh_ffn.write(f">{new_id} {start}-{end} {tag_name}\n{feature_seq}\n")
-
-def filter_faa_file(annotation_per_contigs, faa_file, out_faa):
-
- protein_fa = pyfastx.Fasta(faa_file)
-
- for contig, features in annotation_per_contigs:
-
- for feature in features:
- new_id = f"{feature['seqname']}.{feature['feature']}_{gene_count[feature['feature']]}"
-
- gff_id = get_tag_value(feature['attribute'], 'ID')
- # ID in gff from prodigal is <contig_number>_<cds_number>
- # seq name from prodigal is <contig_name>_<cds_number>
- cds_number = gff_id.split('_')[-1]
- cds_prodigal_id = f"{contig}_{cds_number}"
- print(cds_prodigal_id, new_id)
- print(protein_fa[cds_prodigal_id])
-
-
-
-
- input()
-
-
-
- yield contig, features
-
def main():
@@ -258,8 +235,6 @@ def main():
logging.info('Merge CDS and RNA annotations by contig.')
annotation_per_contigs = merging_cds_and_rna(unoverlapping_cds_per_contig, contig2rnas)
- # annotation_per_contigs = filter_faa_file(annotation_per_contigs, faa_file, out_faa)
-
logging.info(f'Writting CDS and RNA annotations to gff file: {output_gff}.')
writing_features_to_gff_ffn_faa(annotation_per_contigs, output_gff, fna_file, out_ffn, faa_file, out_faa)
--
GitLab
From 6e7f50079ee2fd69f61500c0517ba54fb7ea0c40 Mon Sep 17 00:00:00 2001
From: Jean Mainguy <jean.mainguy@inra.fr>
Date: Thu, 20 Oct 2022 14:40:52 +0200
Subject: [PATCH 017/194] clean script
---
bin/merge_annotations.py | 9 +++++----
1 file changed, 5 insertions(+), 4 deletions(-)
diff --git a/bin/merge_annotations.py b/bin/merge_annotations.py
index 91af848..ac098de 100755
--- a/bin/merge_annotations.py
+++ b/bin/merge_annotations.py
@@ -212,13 +212,14 @@ def main():
rrna_file = args.rrna
cds_file = args.cds
- output_gff = args.gff_output
fna_file = args.contig_seq
+ faa_file = args.faa_file
+
+ out_gff = args.gff_output
out_ffn = args.ffn_output
out_faa = args.faa_output
- faa_file = "prodigal.faa"
logging.info('Parsing rRNA annoations.')
rrna_annotations = parse_gff_file(rrna_file, 'rRNA')
@@ -235,8 +236,8 @@ def main():
logging.info('Merge CDS and RNA annotations by contig.')
annotation_per_contigs = merging_cds_and_rna(unoverlapping_cds_per_contig, contig2rnas)
- logging.info(f'Writting CDS and RNA annotations to gff file: {output_gff}.')
- writing_features_to_gff_ffn_faa(annotation_per_contigs, output_gff, fna_file, out_ffn, faa_file, out_faa)
+ logging.info(f'Writting CDS and RNA annotations to gff file: {out_gff}.')
+ writing_features_to_gff_ffn_faa(annotation_per_contigs, out_gff, fna_file, out_ffn, faa_file, out_faa)
if __name__ == '__main__':
--
GitLab
From 51acc8165a401e498b8d59b3df79c14b4c04a742 Mon Sep 17 00:00:00 2001
From: Jean Mainguy <jean.mainguy@inra.fr>
Date: Thu, 20 Oct 2022 14:45:55 +0200
Subject: [PATCH 018/194] add faa files into merging process
---
modules/merge_annotations.nf | 8 +++++---
subworkflows/04_structural_annot.nf | 2 +-
2 files changed, 6 insertions(+), 4 deletions(-)
diff --git a/modules/merge_annotations.nf b/modules/merge_annotations.nf
index 4cbbd82..12f37e8 100644
--- a/modules/merge_annotations.nf
+++ b/modules/merge_annotations.nf
@@ -2,14 +2,16 @@ process MERGE_ANNOTATIONS {
tag "${meta.id}"
input:
- tuple val(meta), file(assembly_file), file(cds_gff), file(rrna_gff), file(trna_gff)
+ tuple val(meta), file(assembly_file), file(faa_file), file(cds_gff), file(rrna_gff), file(trna_gff)
output:
+
script:
"""
- merge_annotations.py -c $cds_gff -r $rrna_gff -t $trna_gff -v --contig_seq $assembly_file \
- --ffn_output ${meta.sample}.ffn --gff_output ${meta.sample}.gff
+ merge_annotations.py -c $cds_gff -r $rrna_gff -t $trna_gff -v \
+ --contig_seq $assembly_file --faa_file $faa_file \
+ --ffn_output ${meta.sample}.ffn --gff_output ${meta.sample}.gff --faa_output ${meta.sample}.faa
"""
}
\ No newline at end of file
diff --git a/subworkflows/04_structural_annot.nf b/subworkflows/04_structural_annot.nf
index e420d55..7ce7d5e 100644
--- a/subworkflows/04_structural_annot.nf
+++ b/subworkflows/04_structural_annot.nf
@@ -13,7 +13,7 @@ workflow STEP_04_STRUCTURAL_ANNOT {
TRNASCAN_SE( assembly )
- annotations_ch = assembly.join(PRODIGAL.out.gff).join(BARRNAP.out.gff)
+ annotations_ch = assembly.join(PRODIGAL.out.faa).join(PRODIGAL.out.gff).join(BARRNAP.out.gff)
.join(TRNASCAN_SE.out.gff)
annotations_ch.view()
--
GitLab
From 1f88a818b11c258a09415814daab402cb74d7e5c Mon Sep 17 00:00:00 2001
From: Jean Mainguy <jean.mainguy@inra.fr>
Date: Thu, 20 Oct 2022 17:38:04 +0200
Subject: [PATCH 019/194] Generate report to plot stat in multiqc
---
bin/merge_annotations.py | 21 +++++++++++++++++++--
main.nf | 8 ++++----
modules/merge_annotations.nf | 10 +++++++---
subworkflows/04_structural_annot.nf | 11 ++++-------
4 files changed, 34 insertions(+), 16 deletions(-)
diff --git a/bin/merge_annotations.py b/bin/merge_annotations.py
index ac098de..d18ebd0 100755
--- a/bin/merge_annotations.py
+++ b/bin/merge_annotations.py
@@ -49,6 +49,9 @@ def parse_arguments():
parser.add_argument('--faa_output', help='final faa file with all CDS annotations in amino acid.', default="all_annotation.faa")
+ parser.add_argument('--report_output', help='Prokka report like to be able to show annotations in multiqc.', default="annotation_report.txt")
+
+
parser.add_argument("-v", "--verbose", help="increase output verbosity",
action="store_true")
@@ -154,7 +157,6 @@ def writing_features_to_gff_ffn_faa(annotations_per_contig, out_gff, fna_file, o
contig_fa = pyfastx.Fasta(fna_file)
protein_fa = pyfastx.Fasta(faa_file)
-
with open(out_gff, 'w') as fh_gff, open(out_ffn, 'w') as fh_ffn, open(out_faa, 'w') as fh_faa:
fh_gff.write("##gff-version 3\n")
@@ -194,6 +196,16 @@ def writing_features_to_gff_ffn_faa(annotations_per_contig, out_gff, fna_file, o
fh_ffn.write(f">{new_id} {start}-{end} {tag_name}\n{feature_seq}\n")
+ # building prokka like report for multiqc
+ report = {"organism": "NA",
+ "contigs": len(contig_fa),
+ "bases": contig_fa.size }
+ report.update(gene_count)
+
+ return report
+
+
+
def main():
@@ -219,6 +231,7 @@ def main():
out_gff = args.gff_output
out_ffn = args.ffn_output
out_faa = args.faa_output
+ out_report = args.report_output
logging.info('Parsing rRNA annoations.')
@@ -237,7 +250,11 @@ def main():
annotation_per_contigs = merging_cds_and_rna(unoverlapping_cds_per_contig, contig2rnas)
logging.info(f'Writting CDS and RNA annotations to gff file: {out_gff}.')
- writing_features_to_gff_ffn_faa(annotation_per_contigs, out_gff, fna_file, out_ffn, faa_file, out_faa)
+ report = writing_features_to_gff_ffn_faa(annotation_per_contigs, out_gff, fna_file, out_ffn, faa_file, out_faa)
+
+ with open(out_report, "w") as fl:
+ fl.write(''.join(f"{k}: {v}\n" for k,v in report.items()))
+
if __name__ == '__main__':
diff --git a/main.nf b/main.nf
index 788b67f..44a6850 100644
--- a/main.nf
+++ b/main.nf
@@ -446,11 +446,11 @@ workflow {
if ( !params.stop_at_clean && !params.stop_at_assembly && !params.stop_at_filtering ) {
S04_STRUCTURAL_ANNOT ( ch_assembly )
- // ch_prokka_ffn = S04_STRUCTURAL_ANNOT.out.ffn
- // ch_prokka_faa = S04_STRUCTURAL_ANNOT.out.faa
- // ch_prokka_gff = S04_STRUCTURAL_ANNOT.out.gff
+ ch_prokka_ffn = S04_STRUCTURAL_ANNOT.out.ffn
+ ch_prokka_faa = S04_STRUCTURAL_ANNOT.out.faa
+ ch_prokka_gff = S04_STRUCTURAL_ANNOT.out.gff
// ch_prokka_fna = S04_STRUCTURAL_ANNOT.out.fna
- // ch_prokka_report = S04_STRUCTURAL_ANNOT.out.report
+ ch_prokka_report = S04_STRUCTURAL_ANNOT.out.report
// ch_contigs_and_reads = ch_prokka_fna
// .join(ch_reads, remainder: true)
diff --git a/modules/merge_annotations.nf b/modules/merge_annotations.nf
index 12f37e8..c6481f6 100644
--- a/modules/merge_annotations.nf
+++ b/modules/merge_annotations.nf
@@ -1,17 +1,21 @@
process MERGE_ANNOTATIONS {
+ publishDir "${params.outdir}/04_structural_annot/${meta.id}/", mode: 'copy'
tag "${meta.id}"
input:
tuple val(meta), file(assembly_file), file(faa_file), file(cds_gff), file(rrna_gff), file(trna_gff)
output:
-
-
+ tuple val(meta), file("${meta.id}.gff"), emit: gff
+ tuple val(meta), file("${meta.id}.ffn"), emit: ffn
+ tuple val(meta), file("${meta.id}.faa"), emit: faa
+ path "${meta.id}.txt", emit: report
script:
"""
merge_annotations.py -c $cds_gff -r $rrna_gff -t $trna_gff -v \
--contig_seq $assembly_file --faa_file $faa_file \
- --ffn_output ${meta.sample}.ffn --gff_output ${meta.sample}.gff --faa_output ${meta.sample}.faa
+ --ffn_output ${meta.id}.ffn --gff_output ${meta.id}.gff --faa_output ${meta.id}.faa \
+ --report_output ${meta.id}.txt
"""
}
\ No newline at end of file
diff --git a/subworkflows/04_structural_annot.nf b/subworkflows/04_structural_annot.nf
index 7ce7d5e..e315dbd 100644
--- a/subworkflows/04_structural_annot.nf
+++ b/subworkflows/04_structural_annot.nf
@@ -28,14 +28,11 @@ workflow STEP_04_STRUCTURAL_ANNOT {
ch_software_versions = PRODIGAL.out.v_prodigal.first().mix( BARRNAP.out.v_barrnap.first(),
TRNASCAN_SE.out.v_tRNAscan.first())
- // RENAME_CONTIGS_AND_GENES(PROKKA.out.prokka_results)
-
emit:
- // report = PROKKA.out.report
- // fna = RENAME_CONTIGS_AND_GENES.out.fna
- // ffn = RENAME_CONTIGS_AND_GENES.out.ffn
- // gff = RENAME_CONTIGS_AND_GENES.out.gff
- // faa = RENAME_CONTIGS_AND_GENES.out.faa
+ report = MERGE_ANNOTATIONS.out.report
+ ffn = MERGE_ANNOTATIONS.out.ffn
+ gff = MERGE_ANNOTATIONS.out.gff
+ faa = MERGE_ANNOTATIONS.out.faa
// prot_length = RENAME_CONTIGS_AND_GENES.out.prot_length
software_versions = ch_software_versions
}
\ No newline at end of file
--
GitLab
From f3817cb08528be8c66dd2c1ea12bc1a9b53f4cf0 Mon Sep 17 00:00:00 2001
From: Jean Mainguy <jean.mainguy@inra.fr>
Date: Thu, 20 Oct 2022 18:00:14 +0200
Subject: [PATCH 020/194] update multiqc config with new structural annotation
method
---
assets/hifi_multiqc_config.yaml | 7 ++++++-
assets/sr_multiqc_config.yaml | 8 +++++++-
2 files changed, 13 insertions(+), 2 deletions(-)
diff --git a/assets/hifi_multiqc_config.yaml b/assets/hifi_multiqc_config.yaml
index 422ce27..c90beaa 100644
--- a/assets/hifi_multiqc_config.yaml
+++ b/assets/hifi_multiqc_config.yaml
@@ -52,7 +52,9 @@ module_order:
info: 'This section of the report shows quast results after assembly filtering.'
path_filters:
- '*quast_filtered/*/report.tsv'
- - prokka
+ - prokka:
+ name: 'Structural annotation'
+ info: 'This section of the report shows structural annotations results. CDS are predicted using Prodigal, rRNA using Barrnap and tRNA using tRNAscan-se.'
- featureCounts
- custom_content:
name: 'Binning results'
@@ -85,3 +87,6 @@ table_columns_visible:
FastQC (raw):
percent_duplicates: False
percent_gc: False
+ Structural annotation:
+ organism: False
+
diff --git a/assets/sr_multiqc_config.yaml b/assets/sr_multiqc_config.yaml
index ab04863..18076ef 100644
--- a/assets/sr_multiqc_config.yaml
+++ b/assets/sr_multiqc_config.yaml
@@ -64,7 +64,10 @@ module_order:
info: 'This section reports of deduplicated reads alignement against contigs with bwa.'
path_filters:
- '*.count_reads_on_contigs.flagstat'
- - prokka
+ - prokka:
+ name: 'Structural annotation'
+ info: 'This section of the report shows structural annotations results. CDS are predicted using Prodigal, rRNA using Barrnap and tRNA using tRNAscan-se.'
+ - featureCounts
- featureCounts
- custom_content:
name: 'Binning results'
@@ -95,7 +98,10 @@ featurecounts:
fn: '*.summary'
shared: true
+
table_columns_visible:
FastQC (raw):
percent_duplicates: False
percent_gc: False
+ Structural annotation:
+ organism: False
--
GitLab
From 668244600dbe4feaa9d41da5824953ea3a08e179 Mon Sep 17 00:00:00 2001
From: Jean Mainguy <jean.mainguy@inra.fr>
Date: Thu, 20 Oct 2022 23:16:21 +0200
Subject: [PATCH 021/194] rm prokka process and script
---
bin/Rename_contigs_and_genes.py | 166 --------------------------------
modules/prokka.nf | 48 ---------
2 files changed, 214 deletions(-)
delete mode 100755 bin/Rename_contigs_and_genes.py
delete mode 100644 modules/prokka.nf
diff --git a/bin/Rename_contigs_and_genes.py b/bin/Rename_contigs_and_genes.py
deleted file mode 100755
index 7d0e94d..0000000
--- a/bin/Rename_contigs_and_genes.py
+++ /dev/null
@@ -1,166 +0,0 @@
-#!/usr/bin/env python
-
-"""----------------------------------------------------------------------------------------------------------------------------------------------------------
- Script Name: Rename_contigs_and_genes.py
- Description: Rename contigs and genes in GFF, FAA and FFN
- files generated by PROKKA.
- Input files:
- GFF, FAA and FFN files produced by PROKKA.
- Created By: Celine Noirot and Joanna Fourquet
- Date: 2019-06-12
-----------------------------------------------------------------------------------------------------------------------------------------------------------
-"""
-
-# Metadata
-__author__ = 'Celine Noirot and Joanna Fourquet \
-- Plateforme bioinformatique Toulouse'
-__copyright__ = 'Copyright (C) 2019 INRA'
-__license__ = 'GNU General Public License'
-__version__ = '0.1'
-__email__ = 'support.bioinfo.genotoul@inra.fr'
-__status__ = 'dev'
-
-# Status: dev
-
-# Modules importation
-try:
- import argparse
- from BCBio import GFF
- from Bio.Seq import Seq
- from Bio.SeqRecord import SeqRecord
- from Bio.SeqFeature import SeqFeature, FeatureLocation
- import pprint
- from BCBio.GFF import GFFExaminer
- from Bio import SeqIO
-except ImportError as error:
- print(error)
- exit(1)
-
-# Manage parameters
-parser = argparse.ArgumentParser(description = \
-'Script which rename contigs and genes in GFF, FAA and FFN files generated by PROKKA.')
-parser.add_argument('-f', '--file', required = True, help = \
-'GFF file generated by PROKKA.')
-parser.add_argument('-faa', '--fastaFile', required = True, \
-help = 'Fasta of predicted sequence (aa) generated by PROKKA (FAA).')
-parser.add_argument('-ffn', '--ffnFile', required = True, \
-help = 'Fasta of predicted sequence (nuc) generated by PROKKA (FFN).')
-parser.add_argument('-fna', '--fnaFile', required = True, \
-help = 'Fasta of contigs generated by PROKKA.')
-parser.add_argument('-p', '--prefix', required = True, \
-help = 'Contig name prefix.')
-parser.add_argument('-oGFF', '--outGFFFile', required = True, \
-help = 'Name of output GFF file.')
-parser.add_argument('-oFAA', '--outFAAFile', required = True, \
-help = 'Filename of renamed predicted fasta sequences (aa).')
-parser.add_argument('-oFFN', '--outFFNFile', required = True, \
-help = 'Filename of renamed predicted fasta sequences (nuc).')
-parser.add_argument('-oFNA', '--outFNAFile', required = True, \
-help = 'Filename of renamed contig sequences (nuc).')
-parser.add_argument('-oprottable', '--outProtein', default = "protein_table.csv", \
-help = 'Filename for protein names correspondance table.')
-parser.add_argument('-oconttable', '--outContig', default = "contig_table.csv", \
-help = 'Filename for contig names correspondance table.')
-args = parser.parse_args()
-
-
-# Variable names informations:
-# prot: corresponds to proteins
-# ctg: corresponds to contigs
-
-# Variables initialization.
-prot_names = {}
-contig_renames = {}
-ctg_prefix = args.prefix
-prot_prefix = "Prot_"
-to_write = []
-# lecture fna
-#remplissage
-#contig_renames [ald_name]=newname
-#reecriture du fasta
-
-with open(args.fnaFile, "r") as fnaFile,\
- open(args.outFNAFile, "w") as outFNA_handle:
- for record in SeqIO.parse(fnaFile, "fasta"):
- try :
- old_ctg_name = record.id
- new_ctg_name = ctg_prefix + "_c" + old_ctg_name.split("_")[-1]
- contig_renames[old_ctg_name] = new_ctg_name
- record.id = contig_renames[old_ctg_name]
- record.description = record.description.replace(old_ctg_name,"")
- SeqIO.write(record, outFNA_handle, "fasta")
- except :
- print ("Warning FNA file : contig " + record.id + " discarded, no new name defined")
- pass
-
-with open(args.file) as gffFile,\
- open(args.outGFFFile, "w") as out_handle,\
- open(args.outProtein, "w") as fh_prot_table,\
- open(args.outContig, "w") as fh_cont_table,\
- open(args.outContig + ".sed", "w") as fh_cont_sed:
- for rec in GFF.parse(gffFile):
- # Access to contig id
- old_ctg_name = rec.id
- new_ctg_name = contig_renames[old_ctg_name]
- rec.id = new_ctg_name
-
- fh_cont_table.write(old_ctg_name + "\t" + new_ctg_name + "\n")
- fh_cont_sed.write("s/" + old_ctg_name + "/" + new_ctg_name + "/\n")
- # Access to features
- for f_index,feature in enumerate(rec.features):
- if(not(feature.qualifiers['source'][0].startswith("minced"))):
- #Generate correspondance
- old_prot_name = feature.qualifiers['ID'][0].replace("_gene","")
- prot_number = old_prot_name.split("_")[-1]
-
- subfeat_types = {subfeat.type for subfeat in feature.sub_features}
- assert len(subfeat_types) == 1, f'Subfeature have different types {subfeat_types}'
- subfeat_type = subfeat_types.pop()
-
-
- new_prot_name = f"{new_ctg_name}.{subfeat_type}_{prot_number}"
- prot_names[old_prot_name] = new_prot_name
- fh_prot_table.write(old_prot_name + "\t" + new_prot_name + "\n")
-
- #Initialize field of "gene" feature (the parent)
- rec.features[f_index].qualifiers["ID"] = new_prot_name + "_gene"
- rec.features[f_index].qualifiers["locus_tag"] = [new_prot_name]
-
- #Annotations (not prokka lines) are in sub_features
- for fsub_index,sub_feature in enumerate(feature.sub_features):
- # Update ID
- rec.features[f_index].sub_features[fsub_index].qualifiers["ID"] = new_prot_name
- rec.features[f_index].sub_features[fsub_index].qualifiers["Parent"] = []
- rec.features[f_index].sub_features[fsub_index].qualifiers["locus_tag"] = [new_prot_name]
- rec.features[f_index].sub_features[fsub_index].qualifiers["protein_id"] = [new_prot_name]
- to_write.append(rec)
-
- #Write only one time
- #print (to_write)
- GFF.write(to_write, out_handle)
-
-
-with open(args.fastaFile, "r") as handle,\
- open(args.outFAAFile, "w") as outFasta_handle:
- for record in SeqIO.parse(handle, "fasta"):
- try :
- old = record.id
- record.id = prot_names[record.id]
- record.description = record.description.replace(old + " ","")
- SeqIO.write(record, outFasta_handle, "fasta")
- except :
- print ("Warning FAA file : protein " + record.id + " discarded, no new name defined")
- pass
-
-
-with open(args.ffnFile, "r") as handle,\
- open(args.outFFNFile, "w") as outFFN_handle:
- for record in SeqIO.parse(handle, "fasta"):
- try :
- old = record.id
- record.id = prot_names[record.id]
- record.description = record.description.replace(old + " ","")
- SeqIO.write(record, outFFN_handle, "fasta")
- except :
- print ("Warning FFN file : protein " + record.id + " discarded, no new name defined")
- pass
diff --git a/modules/prokka.nf b/modules/prokka.nf
deleted file mode 100644
index 6fbd538..0000000
--- a/modules/prokka.nf
+++ /dev/null
@@ -1,48 +0,0 @@
-process PROKKA {
- tag "${meta.id}"
-
- input:
- tuple val(meta), file(assembly_file)
-
- output:
- tuple val(meta), path("PROKKA_${meta.id}"), emit: prokka_results
- path "PROKKA_${meta.id}/${meta.id}.txt", emit: report
- path "v_prokka.txt", emit: v_prokka
-
- script:
- """
- prokka --metagenome --noanno --rawproduct --outdir PROKKA_${meta.id} --prefix ${meta.id} ${assembly_file} --centre X --compliant --cpus ${task.cpus}
- rm PROKKA_${meta.id}/*.gbk
- gt gff3validator PROKKA_${meta.id}/${meta.id}.gff
-
- prokka -v &> v_prokka.txt
- """
-}
-
-process RENAME_CONTIGS_AND_GENES {
- tag "${meta.id}"
- publishDir "${params.outdir}/04_structural_annot", mode: 'copy'
- label 'PYTHON'
-
- input:
- tuple val(meta), path(prokka_results)
-
- output:
- tuple val(meta), path("${meta.id}.annotated.fna"), emit: fna
- tuple val(meta), path("${meta.id}.annotated.ffn"), emit: ffn
- tuple val(meta), path("${meta.id}.annotated.faa"), emit: faa
- tuple val(meta), path("${meta.id}.annotated.gff"), emit: gff
- tuple val(meta), path("${meta.id}_prot.len"), emit: prot_length
-
- script:
- """
- grep "^gnl" ${prokka_results}/${meta.id}.gff > ${meta.id}_only_gnl.gff
-
- Rename_contigs_and_genes.py -f ${meta.id}_only_gnl.gff -faa ${prokka_results}/${meta.id}.faa \
- -ffn ${prokka_results}/${meta.id}.ffn -fna ${prokka_results}/${meta.id}.fna \
- -p ${meta.id} -oGFF ${meta.id}.annotated.gff -oFAA ${meta.id}.annotated.faa \
- -oFFN ${meta.id}.annotated.ffn -oFNA ${meta.id}.annotated.fna
-
- samtools faidx ${meta.id}.annotated.faa; cut -f 1,2 ${meta.id}.annotated.faa.fai > ${meta.id}_prot.len
- """
-}
--
GitLab
From c6f2778c60764647bbee7733224f4be486bdfac4 Mon Sep 17 00:00:00 2001
From: Jean Mainguy <jean.mainguy@inra.fr>
Date: Thu, 20 Oct 2022 23:18:32 +0200
Subject: [PATCH 022/194] rm conf of removed process
---
conf/base.config | 7 -------
conf/test.config | 7 -------
2 files changed, 14 deletions(-)
diff --git a/conf/base.config b/conf/base.config
index fd060b8..a94490b 100644
--- a/conf/base.config
+++ b/conf/base.config
@@ -58,13 +58,6 @@ process {
memory = { 8.GB * task.attempt }
cpus = 4
}
- withName: PROKKA {
- memory = { 45.GB * task.attempt }
- cpus = 8
- }
- withName: RENAME_CONTIGS_AND_GENES {
- memory = { 20.GB * task.attempt }
- }
withLabel: CD_HIT {
memory = { 50.GB * task.attempt }
cpus = 16
diff --git a/conf/test.config b/conf/test.config
index 710b959..ae50b57 100644
--- a/conf/test.config
+++ b/conf/test.config
@@ -49,13 +49,6 @@ process {
memory = { 1.GB * task.attempt }
cpus = 2
}
- withName: PROKKA {
- memory = { 1.GB * task.attempt }
- cpus = 1
- }
- withName: RENAME_CONTIGS_AND_GENES {
- memory = { 1.GB * task.attempt }
- }
withLabel: CD_HIT {
memory = { 16.GB * task.attempt }
cpus = 2
--
GitLab
From 1092e2592f39c7b93f67fb4a3ef2dee032e223b1 Mon Sep 17 00:00:00 2001
From: Jean Mainguy <jean.mainguy@inra.fr>
Date: Thu, 20 Oct 2022 23:38:45 +0200
Subject: [PATCH 023/194] simple script to rename contigs with sample and
contig number
---
modules/rename_contigs.nf | 0
1 file changed, 0 insertions(+), 0 deletions(-)
create mode 100644 modules/rename_contigs.nf
diff --git a/modules/rename_contigs.nf b/modules/rename_contigs.nf
new file mode 100644
index 0000000..e69de29
--
GitLab
From bbf5ec9720f9ddc4e3fe6cca3ae4b185d91bec56 Mon Sep 17 00:00:00 2001
From: Jean Mainguy <jean.mainguy@inra.fr>
Date: Thu, 20 Oct 2022 23:39:13 +0200
Subject: [PATCH 024/194] make script exec
---
bin/rename_contigs.py | 71 +++++++++++++++++++++++++++++++++++++++++++
1 file changed, 71 insertions(+)
create mode 100755 bin/rename_contigs.py
diff --git a/bin/rename_contigs.py b/bin/rename_contigs.py
new file mode 100755
index 0000000..0734c9c
--- /dev/null
+++ b/bin/rename_contigs.py
@@ -0,0 +1,71 @@
+#!/usr/bin/env python3
+
+"""
+Rename assembly contigs.
+
+Rename contig as <sample_name>_c<contig_number>
+
+:Example:
+rename_contigs.py -h
+"""
+
+# Metadata
+__author__ = 'Mainguy Jean - Plateforme bioinformatique Toulouse'
+__copyright__ = 'Copyright (C) 2020 INRAE'
+__license__ = 'GNU General Public License'
+__version__ = '0.1'
+__email__ = 'support.bioinfo.genotoul@inra.fr'
+__status__ = 'dev'
+
+
+from argparse import ArgumentParser, ArgumentDefaultsHelpFormatter, FileType
+import logging
+import gzip
+import csv
+from collections import defaultdict
+import pyfastx
+from Bio.Seq import Seq
+
+
+def parse_arguments():
+ """Parse script arguments."""
+ parser = ArgumentParser(description="...",
+ formatter_class=ArgumentDefaultsHelpFormatter)
+
+ parser.add_argument('-s', '--sample', help='Sample name used to rename contigs.', required=True)
+
+ parser.add_argument('-i', '--fna_file', help='Original fasta file of contigs.', required=True)
+
+ parser.add_argument('-o', '--out_fna', help='Output fasta file with renamed contigs.', required=True)
+
+ parser.add_argument("-v", "--verbose", help="increase output verbosity",
+ action="store_true")
+
+ args = parser.parse_args()
+ return args
+
+
+
+def main():
+
+ args = parse_arguments()
+
+ if args.verbose:
+ logging.basicConfig(format="%(levelname)s: %(message)s", level=logging.DEBUG)
+ logging.info('Mode verbose ON')
+
+ else:
+ logging.basicConfig(format="%(levelname)s: %(message)s")
+
+ sample = args.sample
+ fna_file = args.fna_file
+ out_fna = args.out_fna
+
+
+ logging.info(f'Writting renamed fasta file in {out_fna}')
+ with open(out_fna, "w") as fh_fna:
+ for i, (_, seq) in enumerate(pyfastx.Fasta(fna_file, build_index=False)):
+ fh_fna.write(f'>{sample}_c{i+1}\n{seq}\n')
+
+if __name__ == '__main__':
+ main()
\ No newline at end of file
--
GitLab
From 52941577a0ee4e4bd8e5c23039ef4c25aa471a53 Mon Sep 17 00:00:00 2001
From: Jean Mainguy <jean.mainguy@inra.fr>
Date: Thu, 20 Oct 2022 23:42:47 +0200
Subject: [PATCH 025/194] call RENAME_CONTIGS process
---
modules/assembly.nf | 17 +++++++++++++++++
1 file changed, 17 insertions(+)
diff --git a/modules/assembly.nf b/modules/assembly.nf
index b8f9d35..41db2c5 100644
--- a/modules/assembly.nf
+++ b/modules/assembly.nf
@@ -107,3 +107,20 @@ process METAFLYE {
"""
}
+process RENAME_CONTIGS {
+ tag "${meta.id}"
+ publishDir "${params.outdir}/02_assembly/", mode: 'copy'
+ label 'PYTHON'
+
+ input:
+ tuple val(meta), path(assembly)
+
+ output:
+ tuple val(meta), path("${meta.id}.fna"), emit: fna
+
+ script:
+ """
+ rename_contigs.py --sample ${meta.id} --fna_file $assembly --out_fna ${meta.id}.fna -v
+
+ """
+}
\ No newline at end of file
--
GitLab
From faec234a8f1a5d6c7e324dfc6723c758d713bd3c Mon Sep 17 00:00:00 2001
From: Jean Mainguy <jean.mainguy@inra.fr>
Date: Thu, 20 Oct 2022 23:50:28 +0200
Subject: [PATCH 026/194] call RENAME_CONTIGS in 02_assembly
---
modules/rename_contigs.nf | 0
subworkflows/02_assembly.nf | 9 +++++----
2 files changed, 5 insertions(+), 4 deletions(-)
delete mode 100644 modules/rename_contigs.nf
diff --git a/modules/rename_contigs.nf b/modules/rename_contigs.nf
deleted file mode 100644
index e69de29..0000000
diff --git a/subworkflows/02_assembly.nf b/subworkflows/02_assembly.nf
index 8411728..0101234 100644
--- a/subworkflows/02_assembly.nf
+++ b/subworkflows/02_assembly.nf
@@ -1,4 +1,4 @@
-include { METASPADES; MEGAHIT; HIFIASM_META; METAFLYE } from '../modules/assembly'
+include { METASPADES; MEGAHIT; HIFIASM_META; METAFLYE; RENAME_CONTIGS } from '../modules/assembly'
include { QUAST as ASSEMBLY_QUAST} from '../modules/metaquast'
include { READS_DEDUPLICATION } from '../modules/reads_deduplication'
include { MINIMAP2 } from '../modules/read_alignment'
@@ -47,7 +47,8 @@ workflow STEP_02_ASSEMBLY {
}
}
- ASSEMBLY_QUAST( ch_assembly,"02_assembly/quast_primary")
+ ch_assembly_renamed = RENAME_CONTIGS(ch_assembly)
+ ASSEMBLY_QUAST( ch_assembly_renamed,"02_assembly/quast_primary")
ch_assembly_report = ASSEMBLY_QUAST.out.report
ch_quast_v = ASSEMBLY_QUAST.out.v_quast
@@ -57,7 +58,7 @@ workflow STEP_02_ASSEMBLY {
ch_flagstat = Channel.empty()
ch_reads = reads
- ch_contigs_and_reads = ch_assembly.join(ch_reads, remainder: true)
+ ch_contigs_and_reads = ch_assembly_renamed.join(ch_reads, remainder: true)
if ( params.type.toUpperCase() == "SR" ) {
READS_DEDUPLICATION ( ch_contigs_and_reads )
ch_dedup = READS_DEDUPLICATION.out.dedup
@@ -88,7 +89,7 @@ workflow STEP_02_ASSEMBLY {
emit:
- assembly = ch_assembly
+ assembly = ch_assembly_renamed
dedup = ch_dedup
idxstats = ch_idxstats
flagstat = ch_flagstat
--
GitLab
From 6cf92e7e3813662c4770c23b8643a5381a6a3a51 Mon Sep 17 00:00:00 2001
From: Jean Mainguy <jean.mainguy@inra.fr>
Date: Mon, 17 Oct 2022 14:57:10 +0200
Subject: [PATCH 027/194] Add prodigal module #163
---
modules/prodigal.nf | 18 ++++++++++++++++++
1 file changed, 18 insertions(+)
create mode 100644 modules/prodigal.nf
diff --git a/modules/prodigal.nf b/modules/prodigal.nf
new file mode 100644
index 0000000..1a9946c
--- /dev/null
+++ b/modules/prodigal.nf
@@ -0,0 +1,18 @@
+process PRODIGAL {
+ tag "${meta.id}"
+
+ input:
+ tuple val(meta), file(assembly_file)
+
+ output:
+ tuple val(meta), path("PROKKA_${meta.id}"), emit: prokka_results
+ path "v_prodigal.txt", emit: v_prokka
+
+ script:
+ """
+ prodigal -i ${assembly_file} -c -p meta -f gff -a prodigal.faa -o prodigal.gff
+
+ prodigal -v 2> v_prodigal.txt
+
+ """
+}
\ No newline at end of file
--
GitLab
From 3219ec1d03a7e37cf6336c882a559183c8438996 Mon Sep 17 00:00:00 2001
From: Jean Mainguy <jean.mainguy@inra.fr>
Date: Mon, 17 Oct 2022 15:51:34 +0200
Subject: [PATCH 028/194] Add barrnap module see issue #163
---
modules/barnap.nf | 18 ++++++++++++++++++
1 file changed, 18 insertions(+)
create mode 100644 modules/barnap.nf
diff --git a/modules/barnap.nf b/modules/barnap.nf
new file mode 100644
index 0000000..0b88b76
--- /dev/null
+++ b/modules/barnap.nf
@@ -0,0 +1,18 @@
+process PRODIGAL {
+ tag "${meta.id}"
+
+ input:
+ tuple val(meta), file(assembly_file)
+
+ output:
+ tuple val(meta), path("barrnap.gff"), emit: barrnap_gff
+ path "v_barrnap.txt", emit: v_barrnap
+
+ script:
+ """
+ barrnap -o barrnap.gff --threads ${task.cpus} ${assembly_file}
+
+ barrnap --version 2> v_barrnap.txt
+
+ """
+}
--
GitLab
From 090dd8fb60b1a15811f3c3090dc8da4d202f281b Mon Sep 17 00:00:00 2001
From: Jean Mainguy <jean.mainguy@inra.fr>
Date: Mon, 17 Oct 2022 15:52:13 +0200
Subject: [PATCH 029/194] Fix typo in prodigal module see issue #163
---
modules/prodigal.nf | 5 +++--
1 file changed, 3 insertions(+), 2 deletions(-)
diff --git a/modules/prodigal.nf b/modules/prodigal.nf
index 1a9946c..be69d07 100644
--- a/modules/prodigal.nf
+++ b/modules/prodigal.nf
@@ -5,8 +5,9 @@ process PRODIGAL {
tuple val(meta), file(assembly_file)
output:
- tuple val(meta), path("PROKKA_${meta.id}"), emit: prokka_results
- path "v_prodigal.txt", emit: v_prokka
+ tuple val(meta), path("prodigal.gff"), emit: prodigal_gff
+ tuple val(meta), path("prodigal.faa"), emit: prodigal_faa
+ path "v_prodigal.txt", emit: v_prodigal
script:
"""
--
GitLab
From 4df1dcc1a25ba4571bc977887fdcb6bfcd5b77a4 Mon Sep 17 00:00:00 2001
From: Jean Mainguy <jean.mainguy@inra.fr>
Date: Mon, 17 Oct 2022 15:55:34 +0200
Subject: [PATCH 030/194] fix module name
---
modules/barnap.nf | 2 +-
1 file changed, 1 insertion(+), 1 deletion(-)
diff --git a/modules/barnap.nf b/modules/barnap.nf
index 0b88b76..302ab6d 100644
--- a/modules/barnap.nf
+++ b/modules/barnap.nf
@@ -1,4 +1,4 @@
-process PRODIGAL {
+process BARRNAP {
tag "${meta.id}"
input:
--
GitLab
From f1ccb099769b60ca5fa95fc2a354468a80111d6b Mon Sep 17 00:00:00 2001
From: Jean Mainguy <jean.mainguy@inra.fr>
Date: Mon, 17 Oct 2022 16:18:15 +0200
Subject: [PATCH 031/194] fix typo in barrnap name module
---
modules/{barnap.nf => barrnap.nf} | 0
1 file changed, 0 insertions(+), 0 deletions(-)
rename modules/{barnap.nf => barrnap.nf} (100%)
diff --git a/modules/barnap.nf b/modules/barrnap.nf
similarity index 100%
rename from modules/barnap.nf
rename to modules/barrnap.nf
--
GitLab
From 17f531e2120bbdf17eec1bd8fc765509ae8eeb98 Mon Sep 17 00:00:00 2001
From: Jean Mainguy <jean.mainguy@inra.fr>
Date: Mon, 17 Oct 2022 17:10:09 +0200
Subject: [PATCH 032/194] structuring step 04 with prodigal + barrnap approach
---
main.nf | 17 +++++++++--------
subworkflows/04_structural_annot.nf | 26 ++++++++++++++++----------
2 files changed, 25 insertions(+), 18 deletions(-)
diff --git a/main.nf b/main.nf
index 229345d..cde0ad1 100644
--- a/main.nf
+++ b/main.nf
@@ -443,17 +443,18 @@ workflow {
if ( !params.stop_at_clean && !params.stop_at_assembly && !params.stop_at_filtering ) {
S04_STRUCTURAL_ANNOT ( ch_assembly )
- ch_prokka_ffn = S04_STRUCTURAL_ANNOT.out.ffn
- ch_prokka_faa = S04_STRUCTURAL_ANNOT.out.faa
- ch_prokka_gff = S04_STRUCTURAL_ANNOT.out.gff
- ch_prokka_fna = S04_STRUCTURAL_ANNOT.out.fna
- ch_prokka_report = S04_STRUCTURAL_ANNOT.out.report
+ // ch_prokka_ffn = S04_STRUCTURAL_ANNOT.out.ffn
+ // ch_prokka_faa = S04_STRUCTURAL_ANNOT.out.faa
+ // ch_prokka_gff = S04_STRUCTURAL_ANNOT.out.gff
+ // ch_prokka_fna = S04_STRUCTURAL_ANNOT.out.fna
+ // ch_prokka_report = S04_STRUCTURAL_ANNOT.out.report
- ch_contigs_and_reads = ch_prokka_fna
- .join(ch_reads, remainder: true)
- ch_prot_length = S04_STRUCTURAL_ANNOT.out.prot_length
+ // ch_contigs_and_reads = ch_prokka_fna
+ // .join(ch_reads, remainder: true)
+ // ch_prot_length = S04_STRUCTURAL_ANNOT.out.prot_length
ch_software_versions_S04 = S04_STRUCTURAL_ANNOT.out.software_versions
+ // exit 0, 'Exit now... S04_STRUCTURAL_ANNOT being constructed '
}
ch_bam = Channel.empty()
diff --git a/subworkflows/04_structural_annot.nf b/subworkflows/04_structural_annot.nf
index 61bff36..ba3d4fe 100644
--- a/subworkflows/04_structural_annot.nf
+++ b/subworkflows/04_structural_annot.nf
@@ -1,19 +1,25 @@
-include { PROKKA; RENAME_CONTIGS_AND_GENES } from '../modules/prokka'
+include { PRODIGAL } from '../modules/prodigal'
+include { BARRNAP } from '../modules/barrnap'
workflow STEP_04_STRUCTURAL_ANNOT {
take: assembly
main:
- PROKKA( assembly )
- ch_software_versions = PROKKA.out.v_prokka.first()
- RENAME_CONTIGS_AND_GENES(PROKKA.out.prokka_results)
+ PRODIGAL( assembly )
+ BARRNAP( assembly )
+ ch_software_versions = PRODIGAL.out.v_prodigal.first()
+
+
+ ch_software_versions = PRODIGAL.out.v_prodigal.first().mix( BARRNAP.out.v_barrnap.first())
+
+ // RENAME_CONTIGS_AND_GENES(PROKKA.out.prokka_results)
emit:
- report = PROKKA.out.report
- fna = RENAME_CONTIGS_AND_GENES.out.fna
- ffn = RENAME_CONTIGS_AND_GENES.out.ffn
- gff = RENAME_CONTIGS_AND_GENES.out.gff
- faa = RENAME_CONTIGS_AND_GENES.out.faa
- prot_length = RENAME_CONTIGS_AND_GENES.out.prot_length
+ // report = PROKKA.out.report
+ // fna = RENAME_CONTIGS_AND_GENES.out.fna
+ // ffn = RENAME_CONTIGS_AND_GENES.out.ffn
+ // gff = RENAME_CONTIGS_AND_GENES.out.gff
+ // faa = RENAME_CONTIGS_AND_GENES.out.faa
+ // prot_length = RENAME_CONTIGS_AND_GENES.out.prot_length
software_versions = ch_software_versions
}
\ No newline at end of file
--
GitLab
From 40bf8662d360e145d7268ad9d7f03812d7a2ef8b Mon Sep 17 00:00:00 2001
From: Jean Mainguy <jean.mainguy@inra.fr>
Date: Mon, 17 Oct 2022 17:10:43 +0200
Subject: [PATCH 033/194] fix barnnap output
---
modules/barrnap.nf | 2 +-
1 file changed, 1 insertion(+), 1 deletion(-)
diff --git a/modules/barrnap.nf b/modules/barrnap.nf
index 302ab6d..44cbd5e 100644
--- a/modules/barrnap.nf
+++ b/modules/barrnap.nf
@@ -10,7 +10,7 @@ process BARRNAP {
script:
"""
- barrnap -o barrnap.gff --threads ${task.cpus} ${assembly_file}
+ barrnap --threads ${task.cpus} ${assembly_file} > barrnap.gff
barrnap --version 2> v_barrnap.txt
--
GitLab
From 80e61aa9048ec99362ed88b3ce5d836a542b048f Mon Sep 17 00:00:00 2001
From: Jean Mainguy <jean.mainguy@inra.fr>
Date: Wed, 19 Oct 2022 10:35:40 +0200
Subject: [PATCH 034/194] Add trnascan and pyfastx in conda recipe
issue #163
---
env/metagWGS.yml | 4 +++-
1 file changed, 3 insertions(+), 1 deletion(-)
diff --git a/env/metagWGS.yml b/env/metagWGS.yml
index 920422d..3708912 100644
--- a/env/metagWGS.yml
+++ b/env/metagWGS.yml
@@ -27,4 +27,6 @@ dependencies:
- subread=2.0.1
- flye=2.9.1
- hifiasm_meta=hamtv0.3
- - plotly
\ No newline at end of file
+ - plotly
+ - trnascan-se=2.0.11
+ - pyfastx
\ No newline at end of file
--
GitLab
From 7ee829d797a6441aba07f25e136b03388fa6f9b3 Mon Sep 17 00:00:00 2001
From: Jean Mainguy <jean.mainguy@inra.fr>
Date: Wed, 19 Oct 2022 11:45:15 +0200
Subject: [PATCH 035/194] Add trnascan_se module issue #163
---
modules/trnascan_se.nf | 19 +++++++++++++++++++
subworkflows/04_structural_annot.nf | 6 +++++-
2 files changed, 24 insertions(+), 1 deletion(-)
create mode 100644 modules/trnascan_se.nf
diff --git a/modules/trnascan_se.nf b/modules/trnascan_se.nf
new file mode 100644
index 0000000..e292e3e
--- /dev/null
+++ b/modules/trnascan_se.nf
@@ -0,0 +1,19 @@
+process TRNASCAN_SE {
+ tag "${meta.id}"
+
+ input:
+ tuple val(meta), file(assembly_file)
+
+ output:
+ tuple val(meta), path("trnascan_se.gff"), emit: trnascan_gff
+ path "v_tRNAscan.txt", emit: v_tRNAscan
+
+ script:
+ """
+ tRNAscan-SE -B --gff trnascan_se.gff --thread ${task.cpus} --stats trnascan_se.log ${assembly_file}
+
+ tRNAscan-SE -h 2> v_tRNAscan.txt
+
+
+ """
+}
diff --git a/subworkflows/04_structural_annot.nf b/subworkflows/04_structural_annot.nf
index ba3d4fe..4e6687b 100644
--- a/subworkflows/04_structural_annot.nf
+++ b/subworkflows/04_structural_annot.nf
@@ -1,5 +1,6 @@
include { PRODIGAL } from '../modules/prodigal'
include { BARRNAP } from '../modules/barrnap'
+include { TRNASCAN_SE } from '../modules/trnascan_se'
workflow STEP_04_STRUCTURAL_ANNOT {
take: assembly
@@ -7,10 +8,13 @@ workflow STEP_04_STRUCTURAL_ANNOT {
main:
PRODIGAL( assembly )
BARRNAP( assembly )
+ TRNASCAN_SE( assembly )
+
ch_software_versions = PRODIGAL.out.v_prodigal.first()
- ch_software_versions = PRODIGAL.out.v_prodigal.first().mix( BARRNAP.out.v_barrnap.first())
+ ch_software_versions = PRODIGAL.out.v_prodigal.first().mix( BARRNAP.out.v_barrnap.first(),
+ TRNASCAN_SE.out.v_tRNAscan.first())
// RENAME_CONTIGS_AND_GENES(PROKKA.out.prokka_results)
--
GitLab
From 6ae87fe3a29afa7580d766d72b461fe1097ce85d Mon Sep 17 00:00:00 2001
From: Jean Mainguy <jean.mainguy@inra.fr>
Date: Wed, 19 Oct 2022 17:48:21 +0200
Subject: [PATCH 036/194] simplify channel name
---
modules/barrnap.nf | 2 +-
modules/prodigal.nf | 4 ++--
modules/trnascan_se.nf | 2 +-
3 files changed, 4 insertions(+), 4 deletions(-)
diff --git a/modules/barrnap.nf b/modules/barrnap.nf
index 44cbd5e..16013a0 100644
--- a/modules/barrnap.nf
+++ b/modules/barrnap.nf
@@ -5,7 +5,7 @@ process BARRNAP {
tuple val(meta), file(assembly_file)
output:
- tuple val(meta), path("barrnap.gff"), emit: barrnap_gff
+ tuple val(meta), path("barrnap.gff"), emit: gff
path "v_barrnap.txt", emit: v_barrnap
script:
diff --git a/modules/prodigal.nf b/modules/prodigal.nf
index be69d07..63bde69 100644
--- a/modules/prodigal.nf
+++ b/modules/prodigal.nf
@@ -5,8 +5,8 @@ process PRODIGAL {
tuple val(meta), file(assembly_file)
output:
- tuple val(meta), path("prodigal.gff"), emit: prodigal_gff
- tuple val(meta), path("prodigal.faa"), emit: prodigal_faa
+ tuple val(meta), path("prodigal.gff"), emit: gff
+ tuple val(meta), path("prodigal.faa"), emit: faa
path "v_prodigal.txt", emit: v_prodigal
script:
diff --git a/modules/trnascan_se.nf b/modules/trnascan_se.nf
index e292e3e..b690c7c 100644
--- a/modules/trnascan_se.nf
+++ b/modules/trnascan_se.nf
@@ -5,7 +5,7 @@ process TRNASCAN_SE {
tuple val(meta), file(assembly_file)
output:
- tuple val(meta), path("trnascan_se.gff"), emit: trnascan_gff
+ tuple val(meta), path("trnascan_se.gff"), emit: gff
path "v_tRNAscan.txt", emit: v_tRNAscan
script:
--
GitLab
From 82b9f4172e919835d844d859fa8a4d4b16338ec4 Mon Sep 17 00:00:00 2001
From: Jean Mainguy <jean.mainguy@inra.fr>
Date: Wed, 19 Oct 2022 18:03:14 +0200
Subject: [PATCH 037/194] Add process and script to merge gff annotations issue
#163
---
bin/merge_annotations.py | 178 ++++++++++++++++++++++++++++
subworkflows/04_structural_annot.nf | 12 ++
2 files changed, 190 insertions(+)
create mode 100755 bin/merge_annotations.py
diff --git a/bin/merge_annotations.py b/bin/merge_annotations.py
new file mode 100755
index 0000000..8d3b4fa
--- /dev/null
+++ b/bin/merge_annotations.py
@@ -0,0 +1,178 @@
+#!/usr/bin/env python3
+
+"""
+Combine structural annotations made from different gff.
+
+In case of collapsing annotations RNA annotation are prefered to CDS annotations.
+
+:Example:
+python template.py -v
+"""
+
+# Metadata
+__author__ = 'Mainguy Jean - Plateforme bioinformatique Toulouse'
+__copyright__ = 'Copyright (C) 2020 INRAE'
+__license__ = 'GNU General Public License'
+__version__ = '0.1'
+__email__ = 'support.bioinfo.genotoul@inra.fr'
+__status__ = 'dev'
+
+
+from argparse import ArgumentParser, ArgumentDefaultsHelpFormatter, FileType
+import logging
+import gzip
+import csv
+from collections import defaultdict
+
+def read_file_and_ignore_hashtag(file_path):
+ proper_open = gzip.open if file_path.endswith('.gz') else open
+ with proper_open(file_path, 'rt') as fl:
+ for line in fl:
+ if line.startswith('#'):
+ continue
+ yield line
+
+def group_annotations_by_contigs(*gff_annotations):
+
+ contig2annotations = defaultdict(list)
+ for annotations in gff_annotations:
+ for annotation in annotations:
+ contig2annotations[annotation['seqname']].append(annotation)
+
+ return contig2annotations
+
+
+def parse_gff_file(gff_file, feature_to_keep=False):
+
+ gff_headers = ("seqname", "_3", "feature", "start", "end", "_2", "strand", "_1", "attribute")
+
+ gff_annotations = csv.DictReader(read_file_and_ignore_hashtag(gff_file),
+ delimiter='\t',
+ fieldnames=gff_headers)
+
+ if feature_to_keep:
+ logging.info(f'Keeping only {feature_to_keep} feature from {gff_file}')
+ gff_annotations = (annotation for annotation in gff_annotations if annotation['feature'] == feature_to_keep)
+
+ return gff_annotations
+
+def parse_arguments():
+ """Parse script arguments."""
+ parser = ArgumentParser(description="...",
+ formatter_class=ArgumentDefaultsHelpFormatter)
+
+ parser.add_argument('-c', '--cds', help='gff file with CDS annotations.', required=True)
+
+ parser.add_argument('-t', '--trna', help='gff file with tRNA annotations.', required=True)
+
+ parser.add_argument('-r', '--rrna', help='gff file with rRNA annotations.', required=True)
+
+ parser.add_argument('-o', '--gff_output', help='final gff file with merged annotations.', required=True)
+
+
+ parser.add_argument("-v", "--verbose", help="increase output verbosity",
+ action="store_true")
+
+ args = parser.parse_args()
+ return args
+
+def remove_overlapping_cds(cds_file, contig2rnas):
+
+ contig_annotations = []
+ current_contig = None
+
+ for cds in parse_gff_file(cds_file, 'CDS'):
+
+ reading_next_contig = cds['seqname'] != current_contig
+
+ if contig_annotations and reading_next_contig:
+
+ yield current_contig, contig_annotations
+ contig_annotations = []
+
+ current_contig = cds['seqname']
+
+ is_overlapping = False
+ for rna in contig2rnas[current_contig]:
+ if (int(rna['end']) < int(cds['start']) or int(rna['start']) > int(cds['end'])):
+ continue
+
+ else: # overlap -> remove cds
+ is_overlapping = True
+ overlap = f"[{max(rna['start'], cds['start'])},{min(rna['end'], cds['end'])}]"
+ rna_info = f"{rna['feature']} [{rna['start']}, {rna['end']}]"
+ cds_info = f"CDS [{cds['start']}, {cds['end']}]"
+ logging.info(f"overlap: {cds_info} overlapping with {rna_info} at {overlap} on contig={current_contig}")
+
+ if not is_overlapping:
+ contig_annotations.append(cds)
+
+ yield current_contig, contig_annotations
+
+def merging_cds_and_rna(cds_per_contig, contig2rnas):
+ contig_processed = []
+ for contig, cds_features in cds_per_contig:
+ contig_processed.append(contig)
+ rna_features = contig2rnas[contig]
+ yield contig, rna_features + cds_features
+
+ # check that all rrna contigs have been processed if not processe them
+ for contig, rnas in contig2rnas.items():
+ if contig not in contig_processed:
+ logging.info(f'{contig} has only rna annotation')
+ yield contig, rnas
+
+def writing_features_to_gff(annotations_per_contig, out_gff):
+
+ with open(out_gff, 'w') as fl:
+
+ fl.write("##gff-version 3\n")
+ for contig, features in annotations_per_contig:
+ logging.info(f"writing {contig} annotations to gff file")
+ fl.write(f"##sequence-region {contig}\n")
+
+ for feature in sorted(features, key=lambda x: int(x['start'])):
+ fl.write('\t'.join(feature.values())+"\n")
+
+def main():
+
+ args = parse_arguments()
+
+ if args.verbose:
+ logging.basicConfig(format="%(levelname)s: %(message)s", level=logging.DEBUG)
+ logging.info('Mode verbose ON')
+
+ else:
+ logging.basicConfig(format="%(levelname)s: %(message)s")
+
+
+ trna_file = args.trna
+ rrna_file = args.rrna
+ cds_file = args.cds
+
+ output_gff = args.gff_output
+
+ logging.info('Parsing rRNA annoations.')
+ rrna_annotations = parse_gff_file(rrna_file, 'rRNA')
+
+ logging.info('Parsing tRNA annoations.')
+ trna_annotations = parse_gff_file(trna_file, 'tRNA')
+
+ logging.info('Grouping RNA annoations by contig.')
+ contig2rnas = group_annotations_by_contigs(trna_annotations, rrna_annotations)
+
+ logging.info('Removing CDS annoations overlapping a RNA annotations.')
+ unoverlapping_cds_per_contig = remove_overlapping_cds(cds_file, contig2rnas)
+
+ logging.info('Merge CDS and RNA annotations by contig.')
+ annotation_per_contigs = merging_cds_and_rna(unoverlapping_cds_per_contig, contig2rnas)
+
+ logging.info(f'Writting CDS and RNA annotations to gff file {output_gff}.')
+ writing_features_to_gff(annotation_per_contigs, output_gff)
+
+
+
+
+
+if __name__ == '__main__':
+ main()
\ No newline at end of file
diff --git a/subworkflows/04_structural_annot.nf b/subworkflows/04_structural_annot.nf
index 4e6687b..a70b215 100644
--- a/subworkflows/04_structural_annot.nf
+++ b/subworkflows/04_structural_annot.nf
@@ -1,6 +1,8 @@
include { PRODIGAL } from '../modules/prodigal'
include { BARRNAP } from '../modules/barrnap'
include { TRNASCAN_SE } from '../modules/trnascan_se'
+include { MERGE_ANNOTATIONS } from '../modules/merge_annotations'
+
workflow STEP_04_STRUCTURAL_ANNOT {
take: assembly
@@ -10,6 +12,16 @@ workflow STEP_04_STRUCTURAL_ANNOT {
BARRNAP( assembly )
TRNASCAN_SE( assembly )
+
+ annotations_ch = PRODIGAL.out.gff.join(BARRNAP.out.gff)
+ .join(TRNASCAN_SE.out.gff)
+ annotations_ch.view()
+
+
+
+ MERGE_ANNOTATIONS(annotations_ch)
+
+
ch_software_versions = PRODIGAL.out.v_prodigal.first()
--
GitLab
From 6c5e309a427600f1dbfaadacd166e3afe8ffc630 Mon Sep 17 00:00:00 2001
From: Jean Mainguy <jean.mainguy@inra.fr>
Date: Wed, 19 Oct 2022 19:01:45 +0200
Subject: [PATCH 038/194] add ffn output on top of gff output
---
bin/merge_annotations.py | 51 +++++++++++++++++++++++++----
subworkflows/04_structural_annot.nf | 2 +-
2 files changed, 45 insertions(+), 8 deletions(-)
diff --git a/bin/merge_annotations.py b/bin/merge_annotations.py
index 8d3b4fa..91ebdb0 100755
--- a/bin/merge_annotations.py
+++ b/bin/merge_annotations.py
@@ -23,6 +23,8 @@ import logging
import gzip
import csv
from collections import defaultdict
+import pyfastx
+
def read_file_and_ignore_hashtag(file_path):
proper_open = gzip.open if file_path.endswith('.gz') else open
@@ -67,7 +69,11 @@ def parse_arguments():
parser.add_argument('-r', '--rrna', help='gff file with rRNA annotations.', required=True)
- parser.add_argument('-o', '--gff_output', help='final gff file with merged annotations.', required=True)
+ parser.add_argument('--gff_output', help='final gff file with all annotations.', default="all_annotation.gff")
+ parser.add_argument('--ffn_output', help='final ffn file with all annotations.', default="all_annotation.ffn")
+
+ parser.add_argument('--contig_seq', help='fasta file of contigs. Needed to extract gene annotations', required=True)
+
parser.add_argument("-v", "--verbose", help="increase output verbosity",
@@ -122,17 +128,44 @@ def merging_cds_and_rna(cds_per_contig, contig2rnas):
logging.info(f'{contig} has only rna annotation')
yield contig, rnas
-def writing_features_to_gff(annotations_per_contig, out_gff):
+def add_new_ID_tag(gff_attributes, new_id):
+ gff_attributes_no_id = ';'.join([attr for attr in gff_attributes.split(';') if attr.split('=')[0] != 'ID'])
+ return f"ID={new_id};{gff_attributes_no_id}"
+
+def get_Name_tag(gff_attributes):
+ for attr in gff_attributes.split(';'):
+ if attr.split('=')[0] == "Name":
+ return attr.split('=')[1]
+ return ''
+
+
+def writing_features_to_gff_and_ffn(annotations_per_contig, out_gff, fna_file, out_ffn):
- with open(out_gff, 'w') as fl:
+ contig_fa = pyfastx.Fasta(fna_file)
+
+
+ with open(out_gff, 'w') as fh_gff, open(out_ffn, 'w') as fh_ffn:
- fl.write("##gff-version 3\n")
+ fh_gff.write("##gff-version 3\n")
for contig, features in annotations_per_contig:
+ gene_count = defaultdict(int)
logging.info(f"writing {contig} annotations to gff file")
- fl.write(f"##sequence-region {contig}\n")
+ fh_gff.write(f"##sequence-region {contig}\n")
for feature in sorted(features, key=lambda x: int(x['start'])):
- fl.write('\t'.join(feature.values())+"\n")
+ gene_count[feature['feature']] += 1
+ new_id = f"{feature['seqname']}.{feature['feature']}_{gene_count[feature['feature']]}"
+
+ feature['attribute'] = add_new_ID_tag(feature['attribute'], new_id)
+
+ tag_name = get_Name_tag(feature['attribute'])
+ fh_gff.write('\t'.join(feature.values())+"\n")
+
+ feature_seq = contig_fa.fetch(contig, [int(feature['start']), int(feature['end'])])
+ fh_ffn.write(f">{new_id} {tag_name}\n")
+ fh_ffn.write(f"{feature_seq}\n")
+
+
def main():
@@ -152,6 +185,9 @@ def main():
output_gff = args.gff_output
+ fna_file = args.contig_seq
+ out_ffn = args.ffn_output
+
logging.info('Parsing rRNA annoations.')
rrna_annotations = parse_gff_file(rrna_file, 'rRNA')
@@ -168,11 +204,12 @@ def main():
annotation_per_contigs = merging_cds_and_rna(unoverlapping_cds_per_contig, contig2rnas)
logging.info(f'Writting CDS and RNA annotations to gff file {output_gff}.')
- writing_features_to_gff(annotation_per_contigs, output_gff)
+ writing_features_to_gff_and_ffn(annotation_per_contigs, output_gff, fna_file, out_ffn)
+
if __name__ == '__main__':
main()
\ No newline at end of file
diff --git a/subworkflows/04_structural_annot.nf b/subworkflows/04_structural_annot.nf
index a70b215..e420d55 100644
--- a/subworkflows/04_structural_annot.nf
+++ b/subworkflows/04_structural_annot.nf
@@ -13,7 +13,7 @@ workflow STEP_04_STRUCTURAL_ANNOT {
TRNASCAN_SE( assembly )
- annotations_ch = PRODIGAL.out.gff.join(BARRNAP.out.gff)
+ annotations_ch = assembly.join(PRODIGAL.out.gff).join(BARRNAP.out.gff)
.join(TRNASCAN_SE.out.gff)
annotations_ch.view()
--
GitLab
From eb8392e1799265f431e3f6e26c8bf9708af03fa6 Mon Sep 17 00:00:00 2001
From: Jean Mainguy <jean.mainguy@inra.fr>
Date: Thu, 20 Oct 2022 09:31:10 +0200
Subject: [PATCH 039/194] process to launch python script to merge gff files
---
modules/merge_annotations.nf | 15 +++++++++++++++
1 file changed, 15 insertions(+)
create mode 100644 modules/merge_annotations.nf
diff --git a/modules/merge_annotations.nf b/modules/merge_annotations.nf
new file mode 100644
index 0000000..4cbbd82
--- /dev/null
+++ b/modules/merge_annotations.nf
@@ -0,0 +1,15 @@
+process MERGE_ANNOTATIONS {
+ tag "${meta.id}"
+
+ input:
+ tuple val(meta), file(assembly_file), file(cds_gff), file(rrna_gff), file(trna_gff)
+
+ output:
+
+
+ script:
+ """
+ merge_annotations.py -c $cds_gff -r $rrna_gff -t $trna_gff -v --contig_seq $assembly_file \
+ --ffn_output ${meta.sample}.ffn --gff_output ${meta.sample}.gff
+ """
+}
\ No newline at end of file
--
GitLab
From 7d1759466fbb08f640ad66a37f0b517eb7c4b674 Mon Sep 17 00:00:00 2001
From: Jean Mainguy <jean.mainguy@inra.fr>
Date: Thu, 20 Oct 2022 13:37:32 +0200
Subject: [PATCH 040/194] fix strand and zero base position when extracting seq
---
bin/merge_annotations.py | 112 +++++++++++++++++++++++++--------------
1 file changed, 73 insertions(+), 39 deletions(-)
diff --git a/bin/merge_annotations.py b/bin/merge_annotations.py
index 91ebdb0..5434cdb 100755
--- a/bin/merge_annotations.py
+++ b/bin/merge_annotations.py
@@ -24,9 +24,36 @@ import gzip
import csv
from collections import defaultdict
import pyfastx
+from Bio.Seq import Seq
+def parse_arguments():
+ """Parse script arguments."""
+ parser = ArgumentParser(description="...",
+ formatter_class=ArgumentDefaultsHelpFormatter)
+
+ parser.add_argument('-c', '--cds', help='gff file with CDS annotations.', required=True)
+
+ parser.add_argument('-t', '--trna', help='gff file with tRNA annotations.', required=True)
+
+ parser.add_argument('-r', '--rrna', help='gff file with rRNA annotations.', required=True)
+
+ parser.add_argument('--gff_output', help='final gff file with all annotations.', default="all_annotation.gff")
+
+ parser.add_argument('--ffn_output', help='final ffn file with all annotations.', default="all_annotation.ffn")
+
+ parser.add_argument('--faa_output', help='final faa file with all CDS annotations in amino acid.', default="all_annotation.faa")
+
+ parser.add_argument('--contig_seq', help='fasta file of contigs. Needed to extract gene annotations', required=True)
+
+ parser.add_argument("-v", "--verbose", help="increase output verbosity",
+ action="store_true")
+
+ args = parser.parse_args()
+ return args
+
def read_file_and_ignore_hashtag(file_path):
+
proper_open = gzip.open if file_path.endswith('.gz') else open
with proper_open(file_path, 'rt') as fl:
for line in fl:
@@ -58,30 +85,6 @@ def parse_gff_file(gff_file, feature_to_keep=False):
return gff_annotations
-def parse_arguments():
- """Parse script arguments."""
- parser = ArgumentParser(description="...",
- formatter_class=ArgumentDefaultsHelpFormatter)
-
- parser.add_argument('-c', '--cds', help='gff file with CDS annotations.', required=True)
-
- parser.add_argument('-t', '--trna', help='gff file with tRNA annotations.', required=True)
-
- parser.add_argument('-r', '--rrna', help='gff file with rRNA annotations.', required=True)
-
- parser.add_argument('--gff_output', help='final gff file with all annotations.', default="all_annotation.gff")
- parser.add_argument('--ffn_output', help='final ffn file with all annotations.', default="all_annotation.ffn")
-
- parser.add_argument('--contig_seq', help='fasta file of contigs. Needed to extract gene annotations', required=True)
-
-
-
- parser.add_argument("-v", "--verbose", help="increase output verbosity",
- action="store_true")
-
- args = parser.parse_args()
- return args
-
def remove_overlapping_cds(cds_file, contig2rnas):
contig_annotations = []
@@ -116,6 +119,7 @@ def remove_overlapping_cds(cds_file, contig2rnas):
yield current_contig, contig_annotations
def merging_cds_and_rna(cds_per_contig, contig2rnas):
+
contig_processed = []
for contig, cds_features in cds_per_contig:
contig_processed.append(contig)
@@ -129,21 +133,22 @@ def merging_cds_and_rna(cds_per_contig, contig2rnas):
yield contig, rnas
def add_new_ID_tag(gff_attributes, new_id):
+
gff_attributes_no_id = ';'.join([attr for attr in gff_attributes.split(';') if attr.split('=')[0] != 'ID'])
return f"ID={new_id};{gff_attributes_no_id}"
-def get_Name_tag(gff_attributes):
- for attr in gff_attributes.split(';'):
- if attr.split('=')[0] == "Name":
+
+def get_tag_value(gff_attribute, tag):
+
+ for attr in gff_attribute.split(';'):
+ if attr.split('=')[0] == tag:
return attr.split('=')[1]
return ''
-
def writing_features_to_gff_and_ffn(annotations_per_contig, out_gff, fna_file, out_ffn):
contig_fa = pyfastx.Fasta(fna_file)
-
with open(out_gff, 'w') as fh_gff, open(out_ffn, 'w') as fh_ffn:
fh_gff.write("##gff-version 3\n")
@@ -158,13 +163,41 @@ def writing_features_to_gff_and_ffn(annotations_per_contig, out_gff, fna_file, o
feature['attribute'] = add_new_ID_tag(feature['attribute'], new_id)
- tag_name = get_Name_tag(feature['attribute'])
+ tag_name = get_tag_value(feature['attribute'], 'Name')
fh_gff.write('\t'.join(feature.values())+"\n")
- feature_seq = contig_fa.fetch(contig, [int(feature['start']), int(feature['end'])])
- fh_ffn.write(f">{new_id} {tag_name}\n")
- fh_ffn.write(f"{feature_seq}\n")
+ # feature_seq = contig_fa.fetch(contig, [int(feature['start']), int(feature['end'])])
+ #
+
+ start_zero, end_zero = int(feature['start'])-1, int(feature['end'])
+
+ if feature['strand'] == "+":
+ feature_seq = contig_fa[contig][start_zero:end_zero].seq
+ else:
+ # reverse complement the sequence
+ feature_seq = contig_fa[contig][start_zero:end_zero].antisense
+
+ fh_ffn.write(f">{new_id} {tag_name}\n{feature_seq}\n")
+
+
+ # if feature['feature'] == "CDS":
+
+ # aa_seq = Seq(feature_seq).translate(table=translation_table, cds=True)
+
+ # fh_faa.write(f'>{new_id}\n{aa_seq}\n')
+
+
+# def filter_faa_file(annotation_per_contigs, faa_file, out_faa):
+
+# protein_fa = pyfastx.Fasta(faa_file)
+# all_names = [set(protein_fa.keys().filter())]
+
+
+# for contig, features in annotation_per_contigs:
+
+
+# yield contig, features
def main():
@@ -187,6 +220,9 @@ def main():
fna_file = args.contig_seq
out_ffn = args.ffn_output
+ out_faa = args.faa_output
+
+ faa_file = "prodigal.faa"
logging.info('Parsing rRNA annoations.')
rrna_annotations = parse_gff_file(rrna_file, 'rRNA')
@@ -203,13 +239,11 @@ def main():
logging.info('Merge CDS and RNA annotations by contig.')
annotation_per_contigs = merging_cds_and_rna(unoverlapping_cds_per_contig, contig2rnas)
- logging.info(f'Writting CDS and RNA annotations to gff file {output_gff}.')
- writing_features_to_gff_and_ffn(annotation_per_contigs, output_gff, fna_file, out_ffn)
-
-
-
-
+ # annotation_per_contigs = filter_faa_file(annotation_per_contigs, faa_file, out_faa)
+ logging.info(f'Writting CDS and RNA annotations to gff file: {output_gff}.')
+ writing_features_to_gff_and_ffn(annotation_per_contigs, output_gff, fna_file, out_ffn) #, out_faa)
+
if __name__ == '__main__':
main()
\ No newline at end of file
--
GitLab
From aef7a100ad95c608c0e20019109b266fa97ac0e3 Mon Sep 17 00:00:00 2001
From: Jean Mainguy <jean.mainguy@inra.fr>
Date: Thu, 20 Oct 2022 14:35:49 +0200
Subject: [PATCH 041/194] add faa file filtering from prodigal
---
bin/merge_annotations.py | 69 +++++++++++++++++++++++++---------------
1 file changed, 44 insertions(+), 25 deletions(-)
diff --git a/bin/merge_annotations.py b/bin/merge_annotations.py
index 5434cdb..e6a5c59 100755
--- a/bin/merge_annotations.py
+++ b/bin/merge_annotations.py
@@ -145,11 +145,13 @@ def get_tag_value(gff_attribute, tag):
return attr.split('=')[1]
return ''
-def writing_features_to_gff_and_ffn(annotations_per_contig, out_gff, fna_file, out_ffn):
+def writing_features_to_gff_ffn_faa(annotations_per_contig, out_gff, fna_file, out_ffn, faa_file, out_faa):
contig_fa = pyfastx.Fasta(fna_file)
+ protein_fa = pyfastx.Fasta(faa_file)
- with open(out_gff, 'w') as fh_gff, open(out_ffn, 'w') as fh_ffn:
+
+ with open(out_gff, 'w') as fh_gff, open(out_ffn, 'w') as fh_ffn, open(out_faa, 'w') as fh_faa:
fh_gff.write("##gff-version 3\n")
for contig, features in annotations_per_contig:
@@ -161,43 +163,60 @@ def writing_features_to_gff_and_ffn(annotations_per_contig, out_gff, fna_file, o
gene_count[feature['feature']] += 1
new_id = f"{feature['seqname']}.{feature['feature']}_{gene_count[feature['feature']]}"
+ start, end = int(feature['start']), int(feature['end'])
+
+ # write faa
+ if feature['feature'] == "CDS":
+ gff_id = get_tag_value(feature['attribute'], 'ID')
+
+ # ID in gff from prodigal is <contig_number>_<cds_number>
+ # seq name in faa from prodigal is <contig_name>_<cds_number>
+ cds_number = gff_id.split('_')[-1]
+ cds_prodigal_id = f"{contig}_{cds_number}"
+ fh_faa.write(f">{new_id} {start}-{end}\n{protein_fa[cds_prodigal_id]}\n")
+
+
+ # Write gff and ffn
feature['attribute'] = add_new_ID_tag(feature['attribute'], new_id)
tag_name = get_tag_value(feature['attribute'], 'Name')
fh_gff.write('\t'.join(feature.values())+"\n")
-
- # feature_seq = contig_fa.fetch(contig, [int(feature['start']), int(feature['end'])])
- #
-
-
- start_zero, end_zero = int(feature['start'])-1, int(feature['end'])
if feature['strand'] == "+":
- feature_seq = contig_fa[contig][start_zero:end_zero].seq
+ feature_seq = contig_fa[contig][start-1:end].seq
else:
- # reverse complement the sequence
- feature_seq = contig_fa[contig][start_zero:end_zero].antisense
+ # minus strand: reverse complement the sequence
+ feature_seq = contig_fa[contig][start-1:end].antisense
- fh_ffn.write(f">{new_id} {tag_name}\n{feature_seq}\n")
+ fh_ffn.write(f">{new_id} {start}-{end} {tag_name}\n{feature_seq}\n")
- # if feature['feature'] == "CDS":
-
- # aa_seq = Seq(feature_seq).translate(table=translation_table, cds=True)
-
- # fh_faa.write(f'>{new_id}\n{aa_seq}\n')
-
+
+def filter_faa_file(annotation_per_contigs, faa_file, out_faa):
-# def filter_faa_file(annotation_per_contigs, faa_file, out_faa):
+ protein_fa = pyfastx.Fasta(faa_file)
-# protein_fa = pyfastx.Fasta(faa_file)
-# all_names = [set(protein_fa.keys().filter())]
+ for contig, features in annotation_per_contigs:
+
+ for feature in features:
+ new_id = f"{feature['seqname']}.{feature['feature']}_{gene_count[feature['feature']]}"
+
+ gff_id = get_tag_value(feature['attribute'], 'ID')
+ # ID in gff from prodigal is <contig_number>_<cds_number>
+ # seq name from prodigal is <contig_name>_<cds_number>
+ cds_number = gff_id.split('_')[-1]
+ cds_prodigal_id = f"{contig}_{cds_number}"
+ print(cds_prodigal_id, new_id)
+ print(protein_fa[cds_prodigal_id])
+
+
+
+
+ input()
-
-# for contig, features in annotation_per_contigs:
-# yield contig, features
+ yield contig, features
def main():
@@ -242,7 +261,7 @@ def main():
# annotation_per_contigs = filter_faa_file(annotation_per_contigs, faa_file, out_faa)
logging.info(f'Writting CDS and RNA annotations to gff file: {output_gff}.')
- writing_features_to_gff_and_ffn(annotation_per_contigs, output_gff, fna_file, out_ffn) #, out_faa)
+ writing_features_to_gff_ffn_faa(annotation_per_contigs, output_gff, fna_file, out_ffn, faa_file, out_faa)
if __name__ == '__main__':
--
GitLab
From e74d1a1f32fa06cf2f977185db8a11fa97961d90 Mon Sep 17 00:00:00 2001
From: Jean Mainguy <jean.mainguy@inra.fr>
Date: Thu, 20 Oct 2022 14:37:34 +0200
Subject: [PATCH 042/194] improve presentation string
---
bin/merge_annotations.py | 41 ++++++++--------------------------------
1 file changed, 8 insertions(+), 33 deletions(-)
diff --git a/bin/merge_annotations.py b/bin/merge_annotations.py
index e6a5c59..91af848 100755
--- a/bin/merge_annotations.py
+++ b/bin/merge_annotations.py
@@ -3,10 +3,11 @@
"""
Combine structural annotations made from different gff.
-In case of collapsing annotations RNA annotation are prefered to CDS annotations.
+In case of collapsing annotations, RNA annotation are prefered to CDS annotations.
+Faa file from prodigal is processed to filter out overlapping sequences.
:Example:
-python template.py -v
+merge_annotations.py -c prodigal.gff -r barrnap.gff -t trnascan_se.gff --contig_seq contigs.fasta --faa_file prodigal.faa
"""
# Metadata
@@ -37,14 +38,17 @@ def parse_arguments():
parser.add_argument('-t', '--trna', help='gff file with tRNA annotations.', required=True)
parser.add_argument('-r', '--rrna', help='gff file with rRNA annotations.', required=True)
+
+ parser.add_argument('--contig_seq', help='fasta file of contigs. Needed to extract gene sequence', required=True)
+
+ parser.add_argument('--faa_file', help='fasta file of protein sequences generated by prodigal.', required=True)
parser.add_argument('--gff_output', help='final gff file with all annotations.', default="all_annotation.gff")
parser.add_argument('--ffn_output', help='final ffn file with all annotations.', default="all_annotation.ffn")
parser.add_argument('--faa_output', help='final faa file with all CDS annotations in amino acid.', default="all_annotation.faa")
-
- parser.add_argument('--contig_seq', help='fasta file of contigs. Needed to extract gene annotations', required=True)
+
parser.add_argument("-v", "--verbose", help="increase output verbosity",
action="store_true")
@@ -191,33 +195,6 @@ def writing_features_to_gff_ffn_faa(annotations_per_contig, out_gff, fna_file, o
fh_ffn.write(f">{new_id} {start}-{end} {tag_name}\n{feature_seq}\n")
-
-def filter_faa_file(annotation_per_contigs, faa_file, out_faa):
-
- protein_fa = pyfastx.Fasta(faa_file)
-
- for contig, features in annotation_per_contigs:
-
- for feature in features:
- new_id = f"{feature['seqname']}.{feature['feature']}_{gene_count[feature['feature']]}"
-
- gff_id = get_tag_value(feature['attribute'], 'ID')
- # ID in gff from prodigal is <contig_number>_<cds_number>
- # seq name from prodigal is <contig_name>_<cds_number>
- cds_number = gff_id.split('_')[-1]
- cds_prodigal_id = f"{contig}_{cds_number}"
- print(cds_prodigal_id, new_id)
- print(protein_fa[cds_prodigal_id])
-
-
-
-
- input()
-
-
-
- yield contig, features
-
def main():
@@ -258,8 +235,6 @@ def main():
logging.info('Merge CDS and RNA annotations by contig.')
annotation_per_contigs = merging_cds_and_rna(unoverlapping_cds_per_contig, contig2rnas)
- # annotation_per_contigs = filter_faa_file(annotation_per_contigs, faa_file, out_faa)
-
logging.info(f'Writting CDS and RNA annotations to gff file: {output_gff}.')
writing_features_to_gff_ffn_faa(annotation_per_contigs, output_gff, fna_file, out_ffn, faa_file, out_faa)
--
GitLab
From daf5224ce69601ed72274df24067ae08b2901c5f Mon Sep 17 00:00:00 2001
From: Jean Mainguy <jean.mainguy@inra.fr>
Date: Thu, 20 Oct 2022 14:40:52 +0200
Subject: [PATCH 043/194] clean script
---
bin/merge_annotations.py | 9 +++++----
1 file changed, 5 insertions(+), 4 deletions(-)
diff --git a/bin/merge_annotations.py b/bin/merge_annotations.py
index 91af848..ac098de 100755
--- a/bin/merge_annotations.py
+++ b/bin/merge_annotations.py
@@ -212,13 +212,14 @@ def main():
rrna_file = args.rrna
cds_file = args.cds
- output_gff = args.gff_output
fna_file = args.contig_seq
+ faa_file = args.faa_file
+
+ out_gff = args.gff_output
out_ffn = args.ffn_output
out_faa = args.faa_output
- faa_file = "prodigal.faa"
logging.info('Parsing rRNA annoations.')
rrna_annotations = parse_gff_file(rrna_file, 'rRNA')
@@ -235,8 +236,8 @@ def main():
logging.info('Merge CDS and RNA annotations by contig.')
annotation_per_contigs = merging_cds_and_rna(unoverlapping_cds_per_contig, contig2rnas)
- logging.info(f'Writting CDS and RNA annotations to gff file: {output_gff}.')
- writing_features_to_gff_ffn_faa(annotation_per_contigs, output_gff, fna_file, out_ffn, faa_file, out_faa)
+ logging.info(f'Writting CDS and RNA annotations to gff file: {out_gff}.')
+ writing_features_to_gff_ffn_faa(annotation_per_contigs, out_gff, fna_file, out_ffn, faa_file, out_faa)
if __name__ == '__main__':
--
GitLab
From 0bc6ba2eb6e1c3a892e790734e84d9d257cfb361 Mon Sep 17 00:00:00 2001
From: Jean Mainguy <jean.mainguy@inra.fr>
Date: Thu, 20 Oct 2022 14:45:55 +0200
Subject: [PATCH 044/194] add faa files into merging process
---
modules/merge_annotations.nf | 8 +++++---
subworkflows/04_structural_annot.nf | 2 +-
2 files changed, 6 insertions(+), 4 deletions(-)
diff --git a/modules/merge_annotations.nf b/modules/merge_annotations.nf
index 4cbbd82..12f37e8 100644
--- a/modules/merge_annotations.nf
+++ b/modules/merge_annotations.nf
@@ -2,14 +2,16 @@ process MERGE_ANNOTATIONS {
tag "${meta.id}"
input:
- tuple val(meta), file(assembly_file), file(cds_gff), file(rrna_gff), file(trna_gff)
+ tuple val(meta), file(assembly_file), file(faa_file), file(cds_gff), file(rrna_gff), file(trna_gff)
output:
+
script:
"""
- merge_annotations.py -c $cds_gff -r $rrna_gff -t $trna_gff -v --contig_seq $assembly_file \
- --ffn_output ${meta.sample}.ffn --gff_output ${meta.sample}.gff
+ merge_annotations.py -c $cds_gff -r $rrna_gff -t $trna_gff -v \
+ --contig_seq $assembly_file --faa_file $faa_file \
+ --ffn_output ${meta.sample}.ffn --gff_output ${meta.sample}.gff --faa_output ${meta.sample}.faa
"""
}
\ No newline at end of file
diff --git a/subworkflows/04_structural_annot.nf b/subworkflows/04_structural_annot.nf
index e420d55..7ce7d5e 100644
--- a/subworkflows/04_structural_annot.nf
+++ b/subworkflows/04_structural_annot.nf
@@ -13,7 +13,7 @@ workflow STEP_04_STRUCTURAL_ANNOT {
TRNASCAN_SE( assembly )
- annotations_ch = assembly.join(PRODIGAL.out.gff).join(BARRNAP.out.gff)
+ annotations_ch = assembly.join(PRODIGAL.out.faa).join(PRODIGAL.out.gff).join(BARRNAP.out.gff)
.join(TRNASCAN_SE.out.gff)
annotations_ch.view()
--
GitLab
From c12388a0bcd7b0161b54820f9f36d731c3b829f1 Mon Sep 17 00:00:00 2001
From: Jean Mainguy <jean.mainguy@inra.fr>
Date: Thu, 20 Oct 2022 17:38:04 +0200
Subject: [PATCH 045/194] Generate report to plot stat in multiqc
---
bin/merge_annotations.py | 21 +++++++++++++++++++--
main.nf | 8 ++++----
modules/merge_annotations.nf | 10 +++++++---
subworkflows/04_structural_annot.nf | 11 ++++-------
4 files changed, 34 insertions(+), 16 deletions(-)
diff --git a/bin/merge_annotations.py b/bin/merge_annotations.py
index ac098de..d18ebd0 100755
--- a/bin/merge_annotations.py
+++ b/bin/merge_annotations.py
@@ -49,6 +49,9 @@ def parse_arguments():
parser.add_argument('--faa_output', help='final faa file with all CDS annotations in amino acid.', default="all_annotation.faa")
+ parser.add_argument('--report_output', help='Prokka report like to be able to show annotations in multiqc.', default="annotation_report.txt")
+
+
parser.add_argument("-v", "--verbose", help="increase output verbosity",
action="store_true")
@@ -154,7 +157,6 @@ def writing_features_to_gff_ffn_faa(annotations_per_contig, out_gff, fna_file, o
contig_fa = pyfastx.Fasta(fna_file)
protein_fa = pyfastx.Fasta(faa_file)
-
with open(out_gff, 'w') as fh_gff, open(out_ffn, 'w') as fh_ffn, open(out_faa, 'w') as fh_faa:
fh_gff.write("##gff-version 3\n")
@@ -194,6 +196,16 @@ def writing_features_to_gff_ffn_faa(annotations_per_contig, out_gff, fna_file, o
fh_ffn.write(f">{new_id} {start}-{end} {tag_name}\n{feature_seq}\n")
+ # building prokka like report for multiqc
+ report = {"organism": "NA",
+ "contigs": len(contig_fa),
+ "bases": contig_fa.size }
+ report.update(gene_count)
+
+ return report
+
+
+
def main():
@@ -219,6 +231,7 @@ def main():
out_gff = args.gff_output
out_ffn = args.ffn_output
out_faa = args.faa_output
+ out_report = args.report_output
logging.info('Parsing rRNA annoations.')
@@ -237,7 +250,11 @@ def main():
annotation_per_contigs = merging_cds_and_rna(unoverlapping_cds_per_contig, contig2rnas)
logging.info(f'Writting CDS and RNA annotations to gff file: {out_gff}.')
- writing_features_to_gff_ffn_faa(annotation_per_contigs, out_gff, fna_file, out_ffn, faa_file, out_faa)
+ report = writing_features_to_gff_ffn_faa(annotation_per_contigs, out_gff, fna_file, out_ffn, faa_file, out_faa)
+
+ with open(out_report, "w") as fl:
+ fl.write(''.join(f"{k}: {v}\n" for k,v in report.items()))
+
if __name__ == '__main__':
diff --git a/main.nf b/main.nf
index cde0ad1..5184c2b 100644
--- a/main.nf
+++ b/main.nf
@@ -443,11 +443,11 @@ workflow {
if ( !params.stop_at_clean && !params.stop_at_assembly && !params.stop_at_filtering ) {
S04_STRUCTURAL_ANNOT ( ch_assembly )
- // ch_prokka_ffn = S04_STRUCTURAL_ANNOT.out.ffn
- // ch_prokka_faa = S04_STRUCTURAL_ANNOT.out.faa
- // ch_prokka_gff = S04_STRUCTURAL_ANNOT.out.gff
+ ch_prokka_ffn = S04_STRUCTURAL_ANNOT.out.ffn
+ ch_prokka_faa = S04_STRUCTURAL_ANNOT.out.faa
+ ch_prokka_gff = S04_STRUCTURAL_ANNOT.out.gff
// ch_prokka_fna = S04_STRUCTURAL_ANNOT.out.fna
- // ch_prokka_report = S04_STRUCTURAL_ANNOT.out.report
+ ch_prokka_report = S04_STRUCTURAL_ANNOT.out.report
// ch_contigs_and_reads = ch_prokka_fna
// .join(ch_reads, remainder: true)
diff --git a/modules/merge_annotations.nf b/modules/merge_annotations.nf
index 12f37e8..c6481f6 100644
--- a/modules/merge_annotations.nf
+++ b/modules/merge_annotations.nf
@@ -1,17 +1,21 @@
process MERGE_ANNOTATIONS {
+ publishDir "${params.outdir}/04_structural_annot/${meta.id}/", mode: 'copy'
tag "${meta.id}"
input:
tuple val(meta), file(assembly_file), file(faa_file), file(cds_gff), file(rrna_gff), file(trna_gff)
output:
-
-
+ tuple val(meta), file("${meta.id}.gff"), emit: gff
+ tuple val(meta), file("${meta.id}.ffn"), emit: ffn
+ tuple val(meta), file("${meta.id}.faa"), emit: faa
+ path "${meta.id}.txt", emit: report
script:
"""
merge_annotations.py -c $cds_gff -r $rrna_gff -t $trna_gff -v \
--contig_seq $assembly_file --faa_file $faa_file \
- --ffn_output ${meta.sample}.ffn --gff_output ${meta.sample}.gff --faa_output ${meta.sample}.faa
+ --ffn_output ${meta.id}.ffn --gff_output ${meta.id}.gff --faa_output ${meta.id}.faa \
+ --report_output ${meta.id}.txt
"""
}
\ No newline at end of file
diff --git a/subworkflows/04_structural_annot.nf b/subworkflows/04_structural_annot.nf
index 7ce7d5e..e315dbd 100644
--- a/subworkflows/04_structural_annot.nf
+++ b/subworkflows/04_structural_annot.nf
@@ -28,14 +28,11 @@ workflow STEP_04_STRUCTURAL_ANNOT {
ch_software_versions = PRODIGAL.out.v_prodigal.first().mix( BARRNAP.out.v_barrnap.first(),
TRNASCAN_SE.out.v_tRNAscan.first())
- // RENAME_CONTIGS_AND_GENES(PROKKA.out.prokka_results)
-
emit:
- // report = PROKKA.out.report
- // fna = RENAME_CONTIGS_AND_GENES.out.fna
- // ffn = RENAME_CONTIGS_AND_GENES.out.ffn
- // gff = RENAME_CONTIGS_AND_GENES.out.gff
- // faa = RENAME_CONTIGS_AND_GENES.out.faa
+ report = MERGE_ANNOTATIONS.out.report
+ ffn = MERGE_ANNOTATIONS.out.ffn
+ gff = MERGE_ANNOTATIONS.out.gff
+ faa = MERGE_ANNOTATIONS.out.faa
// prot_length = RENAME_CONTIGS_AND_GENES.out.prot_length
software_versions = ch_software_versions
}
\ No newline at end of file
--
GitLab
From 6cbc7a4284c229b322eea10f461fc80bbf09886d Mon Sep 17 00:00:00 2001
From: Jean Mainguy <jean.mainguy@inra.fr>
Date: Thu, 20 Oct 2022 18:00:14 +0200
Subject: [PATCH 046/194] update multiqc config with new structural annotation
method
---
assets/hifi_multiqc_config.yaml | 92 +++++++++++++++++++++++++++++++++
assets/multiqc_config.yaml | 7 ++-
2 files changed, 98 insertions(+), 1 deletion(-)
create mode 100644 assets/hifi_multiqc_config.yaml
diff --git a/assets/hifi_multiqc_config.yaml b/assets/hifi_multiqc_config.yaml
new file mode 100644
index 0000000..c90beaa
--- /dev/null
+++ b/assets/hifi_multiqc_config.yaml
@@ -0,0 +1,92 @@
+report_comment: >
+ This report has been generated by the <a href="https://forgemia.inra.fr/genotoul-bioinfo/metagwgs" target="_blank">genotoul-bioinfo/metagwgs</a>
+ analysis pipeline. For information about how to interpret these results, please see the
+ <a href="https://forgemia.inra.fr/genotoul-bioinfo/metagwgs" target="_blank">documentation</a>.
+
+extra_fn_clean_trim:
+ - "hifi_"
+ - 'reads_'
+ - '.count_reads_on_contigs'
+ - '_scaffolds'
+ - '.txt'
+ - '.contigs'
+ - '.sort'
+ - "cleaned_"
+ - "raw_"
+ - '_kept_contigs'
+ - '.no_filter'
+ - '_kaiju_MEM_verbose'
+
+extra_fn_clean_exts:
+ - "_select_contigs_cpm"
+ - '.host_filter'
+
+module_order:
+ - fastqc:
+ name: 'FastQC (raw)'
+ path_filters_exclude:
+ - '*cleaned_*.zip'
+ - samtools:
+ name : 'Reads before host reads filter'
+ info: 'This section reports of the reads alignement against host genome.'
+ path_filters:
+ - '*.no_filter.flagstat'
+ - samtools:
+ name : 'Reads after host reads filter'
+ info: 'This section reports of the cleaned reads alignement against host genome.'
+ path_filters:
+ - '*.host_filter.flagstat'
+ - fastqc:
+ name: 'FastQC (cleaned)'
+ info: 'This section of the report shows FastQC after removing reads mapping the host genome.'
+ path_filters:
+ - '*cleaned_*.zip'
+ - kaiju
+ - quast:
+ name: 'Quast primary assembly'
+ info: 'This section of the report shows quast results after assembly.'
+ path_filters:
+ - '*quast_primary/*/report.tsv'
+ - quast:
+ name: 'Quast filtered assembly'
+ info: 'This section of the report shows quast results after assembly filtering.'
+ path_filters:
+ - '*quast_filtered/*/report.tsv'
+ - prokka:
+ name: 'Structural annotation'
+ info: 'This section of the report shows structural annotations results. CDS are predicted using Prodigal, rRNA using Barrnap and tRNA using tRNAscan-se.'
+ - featureCounts
+ - custom_content:
+ name: 'Binning results'
+ info: 'This section of the report shows quast results after binning the contigs into species genomes'
+ path_filters:
+ - '*mqc.tsv'
+
+report_section_order:
+ stats_table:
+ order: -1000
+ bins_quality:
+ order: -1010
+ bins_quality_count:
+ order: -1020
+ bins_quality_size:
+ order: -1030
+ binning_heatmap:
+ order: -1040
+ software_versions:
+ order: -1050
+
+prokka_fn_snames: True
+prokka_table: True
+
+featurecounts:
+ fn: '*.summary'
+ shared: true
+
+table_columns_visible:
+ FastQC (raw):
+ percent_duplicates: False
+ percent_gc: False
+ Structural annotation:
+ organism: False
+
diff --git a/assets/multiqc_config.yaml b/assets/multiqc_config.yaml
index fc5eb8e..06e9cdd 100644
--- a/assets/multiqc_config.yaml
+++ b/assets/multiqc_config.yaml
@@ -69,11 +69,14 @@ module_order:
path_filters:
- './final_assembly_flagstat/*'
- prokka:
+ name: 'Structural annotation'
+ info: 'This section of the report shows structural annotations results. CDS are predicted using Prodigal, rRNA using Barrnap and tRNA using tRNAscan-se.'
path_filters:
- './prokka_report/*'
- featureCounts:
path_filters:
- './featureCounts_report/*'
+ - '*.count_reads_on_contigs.flagstat'
- custom_content:
name: 'Binning results'
info: 'This section of the report shows quast results after binning the contigs into species genomes'
@@ -106,11 +109,12 @@ featurecounts:
fn: '*.summary'
shared: true
+
table_columns_visible:
FastQC (raw):
percent_duplicates: False
percent_gc: False
- prokka:
+ Structural annotation:
organism: False
Reads alignment on unfiltered assembly:
mapped_passed_pct: True
@@ -141,3 +145,4 @@ table_columns_visible:
# - hide
# show_hide_patterns:
# - ["_R1", "_R2"]
+
--
GitLab
From 7728b4ab4cfd7b992aaa0246dcd71e7a9aea486d Mon Sep 17 00:00:00 2001
From: Jean Mainguy <jean.mainguy@inra.fr>
Date: Thu, 20 Oct 2022 23:16:21 +0200
Subject: [PATCH 047/194] rm prokka process and script
---
bin/Rename_contigs_and_genes.py | 166 --------------------------------
modules/prokka.nf | 48 ---------
2 files changed, 214 deletions(-)
delete mode 100755 bin/Rename_contigs_and_genes.py
delete mode 100644 modules/prokka.nf
diff --git a/bin/Rename_contigs_and_genes.py b/bin/Rename_contigs_and_genes.py
deleted file mode 100755
index 7d0e94d..0000000
--- a/bin/Rename_contigs_and_genes.py
+++ /dev/null
@@ -1,166 +0,0 @@
-#!/usr/bin/env python
-
-"""----------------------------------------------------------------------------------------------------------------------------------------------------------
- Script Name: Rename_contigs_and_genes.py
- Description: Rename contigs and genes in GFF, FAA and FFN
- files generated by PROKKA.
- Input files:
- GFF, FAA and FFN files produced by PROKKA.
- Created By: Celine Noirot and Joanna Fourquet
- Date: 2019-06-12
-----------------------------------------------------------------------------------------------------------------------------------------------------------
-"""
-
-# Metadata
-__author__ = 'Celine Noirot and Joanna Fourquet \
-- Plateforme bioinformatique Toulouse'
-__copyright__ = 'Copyright (C) 2019 INRA'
-__license__ = 'GNU General Public License'
-__version__ = '0.1'
-__email__ = 'support.bioinfo.genotoul@inra.fr'
-__status__ = 'dev'
-
-# Status: dev
-
-# Modules importation
-try:
- import argparse
- from BCBio import GFF
- from Bio.Seq import Seq
- from Bio.SeqRecord import SeqRecord
- from Bio.SeqFeature import SeqFeature, FeatureLocation
- import pprint
- from BCBio.GFF import GFFExaminer
- from Bio import SeqIO
-except ImportError as error:
- print(error)
- exit(1)
-
-# Manage parameters
-parser = argparse.ArgumentParser(description = \
-'Script which rename contigs and genes in GFF, FAA and FFN files generated by PROKKA.')
-parser.add_argument('-f', '--file', required = True, help = \
-'GFF file generated by PROKKA.')
-parser.add_argument('-faa', '--fastaFile', required = True, \
-help = 'Fasta of predicted sequence (aa) generated by PROKKA (FAA).')
-parser.add_argument('-ffn', '--ffnFile', required = True, \
-help = 'Fasta of predicted sequence (nuc) generated by PROKKA (FFN).')
-parser.add_argument('-fna', '--fnaFile', required = True, \
-help = 'Fasta of contigs generated by PROKKA.')
-parser.add_argument('-p', '--prefix', required = True, \
-help = 'Contig name prefix.')
-parser.add_argument('-oGFF', '--outGFFFile', required = True, \
-help = 'Name of output GFF file.')
-parser.add_argument('-oFAA', '--outFAAFile', required = True, \
-help = 'Filename of renamed predicted fasta sequences (aa).')
-parser.add_argument('-oFFN', '--outFFNFile', required = True, \
-help = 'Filename of renamed predicted fasta sequences (nuc).')
-parser.add_argument('-oFNA', '--outFNAFile', required = True, \
-help = 'Filename of renamed contig sequences (nuc).')
-parser.add_argument('-oprottable', '--outProtein', default = "protein_table.csv", \
-help = 'Filename for protein names correspondance table.')
-parser.add_argument('-oconttable', '--outContig', default = "contig_table.csv", \
-help = 'Filename for contig names correspondance table.')
-args = parser.parse_args()
-
-
-# Variable names informations:
-# prot: corresponds to proteins
-# ctg: corresponds to contigs
-
-# Variables initialization.
-prot_names = {}
-contig_renames = {}
-ctg_prefix = args.prefix
-prot_prefix = "Prot_"
-to_write = []
-# lecture fna
-#remplissage
-#contig_renames [ald_name]=newname
-#reecriture du fasta
-
-with open(args.fnaFile, "r") as fnaFile,\
- open(args.outFNAFile, "w") as outFNA_handle:
- for record in SeqIO.parse(fnaFile, "fasta"):
- try :
- old_ctg_name = record.id
- new_ctg_name = ctg_prefix + "_c" + old_ctg_name.split("_")[-1]
- contig_renames[old_ctg_name] = new_ctg_name
- record.id = contig_renames[old_ctg_name]
- record.description = record.description.replace(old_ctg_name,"")
- SeqIO.write(record, outFNA_handle, "fasta")
- except :
- print ("Warning FNA file : contig " + record.id + " discarded, no new name defined")
- pass
-
-with open(args.file) as gffFile,\
- open(args.outGFFFile, "w") as out_handle,\
- open(args.outProtein, "w") as fh_prot_table,\
- open(args.outContig, "w") as fh_cont_table,\
- open(args.outContig + ".sed", "w") as fh_cont_sed:
- for rec in GFF.parse(gffFile):
- # Access to contig id
- old_ctg_name = rec.id
- new_ctg_name = contig_renames[old_ctg_name]
- rec.id = new_ctg_name
-
- fh_cont_table.write(old_ctg_name + "\t" + new_ctg_name + "\n")
- fh_cont_sed.write("s/" + old_ctg_name + "/" + new_ctg_name + "/\n")
- # Access to features
- for f_index,feature in enumerate(rec.features):
- if(not(feature.qualifiers['source'][0].startswith("minced"))):
- #Generate correspondance
- old_prot_name = feature.qualifiers['ID'][0].replace("_gene","")
- prot_number = old_prot_name.split("_")[-1]
-
- subfeat_types = {subfeat.type for subfeat in feature.sub_features}
- assert len(subfeat_types) == 1, f'Subfeature have different types {subfeat_types}'
- subfeat_type = subfeat_types.pop()
-
-
- new_prot_name = f"{new_ctg_name}.{subfeat_type}_{prot_number}"
- prot_names[old_prot_name] = new_prot_name
- fh_prot_table.write(old_prot_name + "\t" + new_prot_name + "\n")
-
- #Initialize field of "gene" feature (the parent)
- rec.features[f_index].qualifiers["ID"] = new_prot_name + "_gene"
- rec.features[f_index].qualifiers["locus_tag"] = [new_prot_name]
-
- #Annotations (not prokka lines) are in sub_features
- for fsub_index,sub_feature in enumerate(feature.sub_features):
- # Update ID
- rec.features[f_index].sub_features[fsub_index].qualifiers["ID"] = new_prot_name
- rec.features[f_index].sub_features[fsub_index].qualifiers["Parent"] = []
- rec.features[f_index].sub_features[fsub_index].qualifiers["locus_tag"] = [new_prot_name]
- rec.features[f_index].sub_features[fsub_index].qualifiers["protein_id"] = [new_prot_name]
- to_write.append(rec)
-
- #Write only one time
- #print (to_write)
- GFF.write(to_write, out_handle)
-
-
-with open(args.fastaFile, "r") as handle,\
- open(args.outFAAFile, "w") as outFasta_handle:
- for record in SeqIO.parse(handle, "fasta"):
- try :
- old = record.id
- record.id = prot_names[record.id]
- record.description = record.description.replace(old + " ","")
- SeqIO.write(record, outFasta_handle, "fasta")
- except :
- print ("Warning FAA file : protein " + record.id + " discarded, no new name defined")
- pass
-
-
-with open(args.ffnFile, "r") as handle,\
- open(args.outFFNFile, "w") as outFFN_handle:
- for record in SeqIO.parse(handle, "fasta"):
- try :
- old = record.id
- record.id = prot_names[record.id]
- record.description = record.description.replace(old + " ","")
- SeqIO.write(record, outFFN_handle, "fasta")
- except :
- print ("Warning FFN file : protein " + record.id + " discarded, no new name defined")
- pass
diff --git a/modules/prokka.nf b/modules/prokka.nf
deleted file mode 100644
index 6fbd538..0000000
--- a/modules/prokka.nf
+++ /dev/null
@@ -1,48 +0,0 @@
-process PROKKA {
- tag "${meta.id}"
-
- input:
- tuple val(meta), file(assembly_file)
-
- output:
- tuple val(meta), path("PROKKA_${meta.id}"), emit: prokka_results
- path "PROKKA_${meta.id}/${meta.id}.txt", emit: report
- path "v_prokka.txt", emit: v_prokka
-
- script:
- """
- prokka --metagenome --noanno --rawproduct --outdir PROKKA_${meta.id} --prefix ${meta.id} ${assembly_file} --centre X --compliant --cpus ${task.cpus}
- rm PROKKA_${meta.id}/*.gbk
- gt gff3validator PROKKA_${meta.id}/${meta.id}.gff
-
- prokka -v &> v_prokka.txt
- """
-}
-
-process RENAME_CONTIGS_AND_GENES {
- tag "${meta.id}"
- publishDir "${params.outdir}/04_structural_annot", mode: 'copy'
- label 'PYTHON'
-
- input:
- tuple val(meta), path(prokka_results)
-
- output:
- tuple val(meta), path("${meta.id}.annotated.fna"), emit: fna
- tuple val(meta), path("${meta.id}.annotated.ffn"), emit: ffn
- tuple val(meta), path("${meta.id}.annotated.faa"), emit: faa
- tuple val(meta), path("${meta.id}.annotated.gff"), emit: gff
- tuple val(meta), path("${meta.id}_prot.len"), emit: prot_length
-
- script:
- """
- grep "^gnl" ${prokka_results}/${meta.id}.gff > ${meta.id}_only_gnl.gff
-
- Rename_contigs_and_genes.py -f ${meta.id}_only_gnl.gff -faa ${prokka_results}/${meta.id}.faa \
- -ffn ${prokka_results}/${meta.id}.ffn -fna ${prokka_results}/${meta.id}.fna \
- -p ${meta.id} -oGFF ${meta.id}.annotated.gff -oFAA ${meta.id}.annotated.faa \
- -oFFN ${meta.id}.annotated.ffn -oFNA ${meta.id}.annotated.fna
-
- samtools faidx ${meta.id}.annotated.faa; cut -f 1,2 ${meta.id}.annotated.faa.fai > ${meta.id}_prot.len
- """
-}
--
GitLab
From d909c03aca2f80309e01de4f38626369549ec413 Mon Sep 17 00:00:00 2001
From: Jean Mainguy <jean.mainguy@inra.fr>
Date: Thu, 20 Oct 2022 23:18:32 +0200
Subject: [PATCH 048/194] rm conf of removed process
---
conf/base.config | 7 -------
conf/test.config | 7 -------
2 files changed, 14 deletions(-)
diff --git a/conf/base.config b/conf/base.config
index fd060b8..a94490b 100644
--- a/conf/base.config
+++ b/conf/base.config
@@ -58,13 +58,6 @@ process {
memory = { 8.GB * task.attempt }
cpus = 4
}
- withName: PROKKA {
- memory = { 45.GB * task.attempt }
- cpus = 8
- }
- withName: RENAME_CONTIGS_AND_GENES {
- memory = { 20.GB * task.attempt }
- }
withLabel: CD_HIT {
memory = { 50.GB * task.attempt }
cpus = 16
diff --git a/conf/test.config b/conf/test.config
index 710b959..ae50b57 100644
--- a/conf/test.config
+++ b/conf/test.config
@@ -49,13 +49,6 @@ process {
memory = { 1.GB * task.attempt }
cpus = 2
}
- withName: PROKKA {
- memory = { 1.GB * task.attempt }
- cpus = 1
- }
- withName: RENAME_CONTIGS_AND_GENES {
- memory = { 1.GB * task.attempt }
- }
withLabel: CD_HIT {
memory = { 16.GB * task.attempt }
cpus = 2
--
GitLab
From a69c4faa43beac99047003b139241f027b6177a8 Mon Sep 17 00:00:00 2001
From: Jean Mainguy <jean.mainguy@inra.fr>
Date: Thu, 20 Oct 2022 23:38:45 +0200
Subject: [PATCH 049/194] simple script to rename contigs with sample and
contig number
---
modules/rename_contigs.nf | 0
1 file changed, 0 insertions(+), 0 deletions(-)
create mode 100644 modules/rename_contigs.nf
diff --git a/modules/rename_contigs.nf b/modules/rename_contigs.nf
new file mode 100644
index 0000000..e69de29
--
GitLab
From fd434081e439fea80cdd12f1f772c40fd8373181 Mon Sep 17 00:00:00 2001
From: Jean Mainguy <jean.mainguy@inra.fr>
Date: Thu, 20 Oct 2022 23:39:13 +0200
Subject: [PATCH 050/194] make script exec
---
bin/rename_contigs.py | 71 +++++++++++++++++++++++++++++++++++++++++++
1 file changed, 71 insertions(+)
create mode 100755 bin/rename_contigs.py
diff --git a/bin/rename_contigs.py b/bin/rename_contigs.py
new file mode 100755
index 0000000..0734c9c
--- /dev/null
+++ b/bin/rename_contigs.py
@@ -0,0 +1,71 @@
+#!/usr/bin/env python3
+
+"""
+Rename assembly contigs.
+
+Rename contig as <sample_name>_c<contig_number>
+
+:Example:
+rename_contigs.py -h
+"""
+
+# Metadata
+__author__ = 'Mainguy Jean - Plateforme bioinformatique Toulouse'
+__copyright__ = 'Copyright (C) 2020 INRAE'
+__license__ = 'GNU General Public License'
+__version__ = '0.1'
+__email__ = 'support.bioinfo.genotoul@inra.fr'
+__status__ = 'dev'
+
+
+from argparse import ArgumentParser, ArgumentDefaultsHelpFormatter, FileType
+import logging
+import gzip
+import csv
+from collections import defaultdict
+import pyfastx
+from Bio.Seq import Seq
+
+
+def parse_arguments():
+ """Parse script arguments."""
+ parser = ArgumentParser(description="...",
+ formatter_class=ArgumentDefaultsHelpFormatter)
+
+ parser.add_argument('-s', '--sample', help='Sample name used to rename contigs.', required=True)
+
+ parser.add_argument('-i', '--fna_file', help='Original fasta file of contigs.', required=True)
+
+ parser.add_argument('-o', '--out_fna', help='Output fasta file with renamed contigs.', required=True)
+
+ parser.add_argument("-v", "--verbose", help="increase output verbosity",
+ action="store_true")
+
+ args = parser.parse_args()
+ return args
+
+
+
+def main():
+
+ args = parse_arguments()
+
+ if args.verbose:
+ logging.basicConfig(format="%(levelname)s: %(message)s", level=logging.DEBUG)
+ logging.info('Mode verbose ON')
+
+ else:
+ logging.basicConfig(format="%(levelname)s: %(message)s")
+
+ sample = args.sample
+ fna_file = args.fna_file
+ out_fna = args.out_fna
+
+
+ logging.info(f'Writting renamed fasta file in {out_fna}')
+ with open(out_fna, "w") as fh_fna:
+ for i, (_, seq) in enumerate(pyfastx.Fasta(fna_file, build_index=False)):
+ fh_fna.write(f'>{sample}_c{i+1}\n{seq}\n')
+
+if __name__ == '__main__':
+ main()
\ No newline at end of file
--
GitLab
From c1f8af42c1d9436d115dc3af56850996dfb6dc59 Mon Sep 17 00:00:00 2001
From: Jean Mainguy <jean.mainguy@inra.fr>
Date: Thu, 20 Oct 2022 23:42:47 +0200
Subject: [PATCH 051/194] call RENAME_CONTIGS process
---
modules/assembly.nf | 17 +++++++++++++++++
1 file changed, 17 insertions(+)
diff --git a/modules/assembly.nf b/modules/assembly.nf
index b8f9d35..41db2c5 100644
--- a/modules/assembly.nf
+++ b/modules/assembly.nf
@@ -107,3 +107,20 @@ process METAFLYE {
"""
}
+process RENAME_CONTIGS {
+ tag "${meta.id}"
+ publishDir "${params.outdir}/02_assembly/", mode: 'copy'
+ label 'PYTHON'
+
+ input:
+ tuple val(meta), path(assembly)
+
+ output:
+ tuple val(meta), path("${meta.id}.fna"), emit: fna
+
+ script:
+ """
+ rename_contigs.py --sample ${meta.id} --fna_file $assembly --out_fna ${meta.id}.fna -v
+
+ """
+}
\ No newline at end of file
--
GitLab
From f8f655cfb5071d8800aa1c9c25b350bc60544854 Mon Sep 17 00:00:00 2001
From: Jean Mainguy <jean.mainguy@inra.fr>
Date: Thu, 20 Oct 2022 23:50:28 +0200
Subject: [PATCH 052/194] call RENAME_CONTIGS in 02_assembly
---
modules/rename_contigs.nf | 0
subworkflows/02_assembly.nf | 9 +++++----
2 files changed, 5 insertions(+), 4 deletions(-)
delete mode 100644 modules/rename_contigs.nf
diff --git a/modules/rename_contigs.nf b/modules/rename_contigs.nf
deleted file mode 100644
index e69de29..0000000
diff --git a/subworkflows/02_assembly.nf b/subworkflows/02_assembly.nf
index 8411728..0101234 100644
--- a/subworkflows/02_assembly.nf
+++ b/subworkflows/02_assembly.nf
@@ -1,4 +1,4 @@
-include { METASPADES; MEGAHIT; HIFIASM_META; METAFLYE } from '../modules/assembly'
+include { METASPADES; MEGAHIT; HIFIASM_META; METAFLYE; RENAME_CONTIGS } from '../modules/assembly'
include { QUAST as ASSEMBLY_QUAST} from '../modules/metaquast'
include { READS_DEDUPLICATION } from '../modules/reads_deduplication'
include { MINIMAP2 } from '../modules/read_alignment'
@@ -47,7 +47,8 @@ workflow STEP_02_ASSEMBLY {
}
}
- ASSEMBLY_QUAST( ch_assembly,"02_assembly/quast_primary")
+ ch_assembly_renamed = RENAME_CONTIGS(ch_assembly)
+ ASSEMBLY_QUAST( ch_assembly_renamed,"02_assembly/quast_primary")
ch_assembly_report = ASSEMBLY_QUAST.out.report
ch_quast_v = ASSEMBLY_QUAST.out.v_quast
@@ -57,7 +58,7 @@ workflow STEP_02_ASSEMBLY {
ch_flagstat = Channel.empty()
ch_reads = reads
- ch_contigs_and_reads = ch_assembly.join(ch_reads, remainder: true)
+ ch_contigs_and_reads = ch_assembly_renamed.join(ch_reads, remainder: true)
if ( params.type.toUpperCase() == "SR" ) {
READS_DEDUPLICATION ( ch_contigs_and_reads )
ch_dedup = READS_DEDUPLICATION.out.dedup
@@ -88,7 +89,7 @@ workflow STEP_02_ASSEMBLY {
emit:
- assembly = ch_assembly
+ assembly = ch_assembly_renamed
dedup = ch_dedup
idxstats = ch_idxstats
flagstat = ch_flagstat
--
GitLab
From db915f01142e804b74e1d180ac0bd99adda55c87 Mon Sep 17 00:00:00 2001
From: Jean Mainguy <jean.mainguy@inra.fr>
Date: Mon, 24 Oct 2022 12:21:20 +0200
Subject: [PATCH 053/194] simplify quast report channel
---
main.nf | 13 ++++++++-----
modules/metaquast.nf | 2 +-
2 files changed, 9 insertions(+), 6 deletions(-)
diff --git a/main.nf b/main.nf
index 5184c2b..fd9fffe 100644
--- a/main.nf
+++ b/main.nf
@@ -404,12 +404,16 @@ workflow {
/////////////////////
S02_ASSEMBLY ( ch_preprocessed_reads, ch_assembly, has_assembly, assembly_tool )
+
ch_assembly = S02_ASSEMBLY.out.assembly
- ch_reads = S02_ASSEMBLY.out.dedup
+ ch_reads = S02_ASSEMBLY.out.reads
+
+
ch_idxstats = S02_ASSEMBLY.out.idxstats
+
ch_unfilter_assembly_flagstat_report = S02_ASSEMBLY.out.flagstat
- ch_quast = S02_ASSEMBLY.out.assembly_report
- ch_quast_before_filter_report = ch_quast.map{ meta, quast_report -> quast_report }
+
+ ch_quast_before_filter_report = S02_ASSEMBLY.out.assembly_report
ch_software_versions_S02 = S02_ASSEMBLY.out.software_versions
}
@@ -429,8 +433,7 @@ workflow {
ch_min_contigs_cpm
)
ch_assembly = S03_FILTERING.out.selected
- ch_quast = S03_FILTERING.out.report
- ch_quast_after_filter_report = ch_quast.map{ meta, quast_report -> quast_report }
+ ch_quast_after_filter_report = S03_FILTERING.out.report
}
diff --git a/modules/metaquast.nf b/modules/metaquast.nf
index dbaa508..dee90d5 100644
--- a/modules/metaquast.nf
+++ b/modules/metaquast.nf
@@ -9,7 +9,7 @@ process QUAST {
output:
path "${outdir}/${meta.id}/*", emit: all
- tuple val(meta), path ("${outdir}/${meta.id}/report.tsv"), emit: report
+ path "${outdir}/${meta.id}/report.tsv", emit: report
path "v_quast.txt", emit: v_quast
script:
--
GitLab
From 28d76a50f041a072f154a5db0443fb2bf6591d35 Mon Sep 17 00:00:00 2001
From: Jean Mainguy <jean.mainguy@inra.fr>
Date: Mon, 24 Oct 2022 14:30:19 +0200
Subject: [PATCH 054/194] clean subworkflow STEP_04_STRUCTURAL_ANNOT
---
subworkflows/04_structural_annot.nf | 6 ------
1 file changed, 6 deletions(-)
diff --git a/subworkflows/04_structural_annot.nf b/subworkflows/04_structural_annot.nf
index e315dbd..edac18e 100644
--- a/subworkflows/04_structural_annot.nf
+++ b/subworkflows/04_structural_annot.nf
@@ -12,19 +12,13 @@ workflow STEP_04_STRUCTURAL_ANNOT {
BARRNAP( assembly )
TRNASCAN_SE( assembly )
-
annotations_ch = assembly.join(PRODIGAL.out.faa).join(PRODIGAL.out.gff).join(BARRNAP.out.gff)
.join(TRNASCAN_SE.out.gff)
- annotations_ch.view()
-
-
MERGE_ANNOTATIONS(annotations_ch)
-
ch_software_versions = PRODIGAL.out.v_prodigal.first()
-
ch_software_versions = PRODIGAL.out.v_prodigal.first().mix( BARRNAP.out.v_barrnap.first(),
TRNASCAN_SE.out.v_tRNAscan.first())
--
GitLab
From 898e8539889162c956047da79d13030824e74e0b Mon Sep 17 00:00:00 2001
From: Jean Mainguy <jean.mainguy@inra.fr>
Date: Mon, 24 Oct 2022 14:34:12 +0200
Subject: [PATCH 055/194] reorganise deduplication process
---
main.nf | 2 +-
modules/reads_deduplication.nf | 43 ++++++++++++++++++++--------------
subworkflows/02_assembly.nf | 20 ++++++++++------
3 files changed, 39 insertions(+), 26 deletions(-)
diff --git a/main.nf b/main.nf
index fd9fffe..bba6ea7 100644
--- a/main.nf
+++ b/main.nf
@@ -407,7 +407,7 @@ workflow {
ch_assembly = S02_ASSEMBLY.out.assembly
ch_reads = S02_ASSEMBLY.out.reads
-
+ ch_bam = S02_ASSEMBLY.out.bam
ch_idxstats = S02_ASSEMBLY.out.idxstats
diff --git a/modules/reads_deduplication.nf b/modules/reads_deduplication.nf
index b64df7c..4731f9b 100644
--- a/modules/reads_deduplication.nf
+++ b/modules/reads_deduplication.nf
@@ -1,38 +1,45 @@
process READS_DEDUPLICATION {
tag "${meta.id}"
- publishDir "${params.outdir}/02_assembly", mode: 'copy', pattern: '*.fastq.gz'
- publishDir "${params.outdir}/02_assembly/logs", mode: 'copy', pattern: '*.idxstats'
- publishDir "${params.outdir}/02_assembly/logs", mode: 'copy', pattern: '*.flagstat'
+ publishDir "${params.outdir}/02_assembly/deduplicated_reads/", mode: 'copy', pattern: '*.fastq.gz'
+ publishDir "${params.outdir}/02_assembly/${meta.id}/", mode: 'copy', pattern: '*.fastq.gz'
input:
tuple val(meta), path(assembly), path(reads)
output:
- tuple val(meta), path("${meta.id}*_dedup.fastq.gz"), emit: dedup
- tuple val(meta), path("${meta.id}.count_reads_on_contigs.idxstats"), emit: idxstats
+ tuple val(meta), path("${meta.id}*_dedup.fastq.gz"), emit: dedup_reads
+ tuple val(meta), path("${meta.id}.bam"), path("${meta.id}.bam.bai"), emit: bam
+
path "${meta.id}_singletons.fastq.gz", emit: singletons
- path "${meta.id}.count_reads_on_contigs.flagstat", emit: flagstat
+
path "v_bwa.txt", emit: v_bwa_mem2
path "v_samtools.txt", emit: v_samtools
script:
"""
- mkdir logs
+ # Align reads against assembly
bwa-mem2 index ${assembly} -p ${assembly}
- bwa-mem2 mem ${assembly} ${reads[0]} ${reads[1]} | samtools view -bS - | samtools sort -n -o ${meta.id}.sort.bam -
- samtools fixmate -m ${meta.id}.sort.bam ${meta.id}.fixmate.bam
- samtools sort -o ${meta.id}.fixmate.positionsort.bam ${meta.id}.fixmate.bam
- samtools markdup -r -S -s -f ${meta.id}.stats ${meta.id}.fixmate.positionsort.bam ${meta.id}.filtered.bam
- samtools index ${meta.id}.filtered.bam
- samtools idxstats ${meta.id}.filtered.bam > ${meta.id}.count_reads_on_contigs.idxstats
- samtools flagstat ${meta.id}.filtered.bam > ${meta.id}.count_reads_on_contigs.flagstat
- samtools sort -n -o ${meta.id}.filtered.sort.bam ${meta.id}.filtered.bam
- samtools fastq -N -s ${meta.id}_singletons.fastq.gz -1 ${meta.id}_R1_dedup.fastq.gz -2 ${meta.id}_R2_dedup.fastq.gz ${meta.id}.filtered.sort.bam
+ bwa-mem2 mem -t ${task.cpus} ${assembly} ${reads[0]} ${reads[1]} | samtools view -@ ${task.cpus} -bS - | samtools sort -@ ${task.cpus} -n -o ${meta.id}.sort.bam -
+
+ # Identify and removed duplicated reads from the bam file
+ samtools fixmate -@ ${task.cpus} -m ${meta.id}.sort.bam ${meta.id}.fixmate.bam
+ samtools sort -@ ${task.cpus} -o ${meta.id}.fixmate.positionsort.bam ${meta.id}.fixmate.bam
+ samtools markdup -@ ${task.cpus} -r -S -s -f ${meta.id}.stats ${meta.id}.fixmate.positionsort.bam ${meta.id}.filtered.bam
+
+ # final bam file without duplicated reads
+ samtools sort -@ ${task.cpus} -o ${meta.id}.bam ${meta.id}.filtered.bam
+ samtools index -@ ${task.cpus} ${meta.id}.bam
+
+ # Get deduplicated reads
+ samtools sort -@ ${task.cpus} -n -o ${meta.id}.filtered.n_sorted.bam ${meta.id}.bam
+ samtools fastq -@ ${task.cpus} -N -s ${meta.id}_singletons.fastq.gz -1 ${meta.id}_R1_dedup.fastq.gz -2 ${meta.id}_R2_dedup.fastq.gz ${meta.id}.filtered.n_sorted.bam
+
+ # clean directory
rm ${meta.id}.sort.bam
rm ${meta.id}.fixmate.bam
rm ${meta.id}.fixmate.positionsort.bam
- rm ${meta.id}.filtered.bam
- rm ${meta.id}.filtered.sort.bam
+ rm ${meta.id}.filtered.n_sorted.bam
+ rm ${meta.id}.filtered.bam
bwa-mem2 version > v_bwa.txt
diff --git a/subworkflows/02_assembly.nf b/subworkflows/02_assembly.nf
index 0101234..6f6141e 100644
--- a/subworkflows/02_assembly.nf
+++ b/subworkflows/02_assembly.nf
@@ -52,26 +52,31 @@ workflow STEP_02_ASSEMBLY {
ch_assembly_report = ASSEMBLY_QUAST.out.report
ch_quast_v = ASSEMBLY_QUAST.out.v_quast
- ch_dedup = Channel.empty()
ch_idxstats = Channel.empty()
- ch_dedup_report = Channel.empty()
ch_flagstat = Channel.empty()
ch_reads = reads
ch_contigs_and_reads = ch_assembly_renamed.join(ch_reads, remainder: true)
if ( params.type.toUpperCase() == "SR" ) {
+
READS_DEDUPLICATION ( ch_contigs_and_reads )
- ch_dedup = READS_DEDUPLICATION.out.dedup
- ch_idxstats = READS_DEDUPLICATION.out.idxstats
- ch_flagstat = READS_DEDUPLICATION.out.flagstat
+
+ ch_reads = READS_DEDUPLICATION.out.dedup_reads
+ ch_bam = READS_DEDUPLICATION.out.bam
+
+ SAMTOOLS(ch_bam, "02_assembly")
+ ch_idxstats = SAMTOOLS.out.sam_idxstat
+ ch_flagstat = SAMTOOLS.out.sam_flagstat
ch_bwa_mem_v = READS_DEDUPLICATION.out.v_bwa_mem2
ch_samtools_v = READS_DEDUPLICATION.out.v_samtools
+
} else {
- ch_dedup = ch_reads
+
if (!params.skip_filtering) {
MINIMAP2(ch_contigs_and_reads,"02_assembly")
ch_bam = MINIMAP2.out.bam
+
SAMTOOLS(ch_bam, "02_assembly")
ch_idxstats = SAMTOOLS.out.sam_idxstat
ch_flagstat = SAMTOOLS.out.sam_flagstat
@@ -90,7 +95,8 @@ workflow STEP_02_ASSEMBLY {
emit:
assembly = ch_assembly_renamed
- dedup = ch_dedup
+ reads = ch_reads
+ bam = ch_bam
idxstats = ch_idxstats
flagstat = ch_flagstat
assembly_report = ch_assembly_report
--
GitLab
From 4cce1264fb5552836f88705efe36c2e1176a4745 Mon Sep 17 00:00:00 2001
From: Jean Mainguy <jean.mainguy@inra.fr>
Date: Mon, 24 Oct 2022 14:35:00 +0200
Subject: [PATCH 056/194] correct typo in multiqc_config
---
assets/multiqc_config.yaml | 2 +-
1 file changed, 1 insertion(+), 1 deletion(-)
diff --git a/assets/multiqc_config.yaml b/assets/multiqc_config.yaml
index 06e9cdd..c24c305 100644
--- a/assets/multiqc_config.yaml
+++ b/assets/multiqc_config.yaml
@@ -75,7 +75,7 @@ module_order:
- './prokka_report/*'
- featureCounts:
path_filters:
- - './featureCounts_report/*'
+ - './featureCounts_report/*'
- '*.count_reads_on_contigs.flagstat'
- custom_content:
name: 'Binning results'
--
GitLab
From 9f2d9a5c97089ecd15fdbf7bcc2becf56180fad9 Mon Sep 17 00:00:00 2001
From: Jean Mainguy <jean.mainguy@inra.fr>
Date: Mon, 24 Oct 2022 14:37:21 +0200
Subject: [PATCH 057/194] rm tax plot made from prot length
---
bin/aln2taxaffi.py | 15 ++++++++-------
main.nf | 4 +---
subworkflows/07_taxonomic_affi.nf | 2 --
3 files changed, 9 insertions(+), 12 deletions(-)
diff --git a/bin/aln2taxaffi.py b/bin/aln2taxaffi.py
index 5c85116..241d836 100755
--- a/bin/aln2taxaffi.py
+++ b/bin/aln2taxaffi.py
@@ -597,13 +597,14 @@ def main():
logging.debug(contig_affi_line)
-
- with open(query_length_file) as fl:
- nb_total_prot = len([line for line in fl])
-
- nb_prot_annotated = len(matches)
- plot_taxonomic_assignment(
- output_name, count_rank_affiliation_protein, count_rank_affiliation_contig, nb_total_prot, nb_prot_annotated, nb_prot_assigned)
+ if query_length_file:
+ logging.debug("Plot taxonomic affiliation using protein lengths.")
+ with open(query_length_file) as fl:
+ nb_total_prot = len([line for line in fl])
+
+ nb_prot_annotated = len(matches)
+ plot_taxonomic_assignment(
+ output_name, count_rank_affiliation_protein, count_rank_affiliation_contig, nb_total_prot, nb_prot_annotated, nb_prot_assigned)
if args.write_top_taxons:
top_taxon_columns = ['contig', ] + main_ranks
diff --git a/main.nf b/main.nf
index bba6ea7..75c9f7b 100644
--- a/main.nf
+++ b/main.nf
@@ -442,7 +442,6 @@ workflow {
ch_prokka_faa = Channel.empty()
ch_prokka_gff = Channel.empty()
ch_prokka_fna = Channel.empty()
- ch_prot_length = Channel.empty()
if ( !params.stop_at_clean && !params.stop_at_assembly && !params.stop_at_filtering ) {
S04_STRUCTURAL_ANNOT ( ch_assembly )
@@ -454,7 +453,6 @@ workflow {
// ch_contigs_and_reads = ch_prokka_fna
// .join(ch_reads, remainder: true)
- // ch_prot_length = S04_STRUCTURAL_ANNOT.out.prot_length
ch_software_versions_S04 = S04_STRUCTURAL_ANNOT.out.software_versions
// exit 0, 'Exit now... S04_STRUCTURAL_ANNOT being constructed '
@@ -487,7 +485,7 @@ workflow {
}
if ( !params.stop_at_clean && !params.stop_at_assembly && !params.stop_at_filtering && !params.stop_at_structural_annot && !params.skip_taxo_affi ) {
- S07_TAXO_AFFI ( ch_taxonomy, ch_m8, ch_sam_coverage, ch_prot_length)
+ S07_TAXO_AFFI ( ch_taxonomy, ch_m8, ch_sam_coverage)
ch_software_versions_S07 = S07_TAXO_AFFI.out.software_versions
}
diff --git a/subworkflows/07_taxonomic_affi.nf b/subworkflows/07_taxonomic_affi.nf
index d1042c0..8eb2640 100644
--- a/subworkflows/07_taxonomic_affi.nf
+++ b/subworkflows/07_taxonomic_affi.nf
@@ -7,10 +7,8 @@ workflow STEP_07_TAXO_AFFI {
taxonomy
diamond_result // channel: [ val(meta), path(diamond_file) ]
sam_coverage // channel: [ val(meta), path(samtools coverage) ]
- prot_length // channel: [ val(meta), path(prot_length) ]
main:
ch_assign_taxo_input = diamond_result.join(sam_coverage, remainder: true)
- .join(prot_length, remainder: true)
ASSIGN_TAXONOMY ( taxonomy, ch_assign_taxo_input )
--
GitLab
From 041d3bb828f4898f904d9b768c0948f6a12fb700 Mon Sep 17 00:00:00 2001
From: Jean Mainguy <jean.mainguy@inra.fr>
Date: Mon, 24 Oct 2022 14:57:00 +0200
Subject: [PATCH 058/194] add new tools in scrape_software_versions used
instead of prokka
---
bin/scrape_software_versions.py | 9 +++++++--
1 file changed, 7 insertions(+), 2 deletions(-)
diff --git a/bin/scrape_software_versions.py b/bin/scrape_software_versions.py
index e04c4d3..4279474 100755
--- a/bin/scrape_software_versions.py
+++ b/bin/scrape_software_versions.py
@@ -23,7 +23,6 @@ regexes = {
'Hifiasm': ['v_hifiasm_meta.txt', r"ha base version: (\S+)"],
'MetaFlye': ['v_metaflye.txt', r"v(\S+)"],
'Quast': ['v_quast.txt', r"QUAST v(\S+)"],
- 'Prokka': ['v_prokka.txt', r"prokka (\S+)"],
'Kaiju': ['v_kaiju.txt', r"Kaiju (\S+)"],
'Samtools': ['v_samtools.txt', r"samtools (\S+)"],
'Eggnog-Mapper': ['v_eggnogmapper.txt', r"emapper-(\S+)"],
@@ -33,7 +32,10 @@ regexes = {
'CheckM2': ['v_checkm2.txt', r"(\S+)"],
'Metawrap': ['v_metawrap.txt', r"metawrap (\S+)"],
'GTDBTK': ['v_gtdbtk.txt', r"...::: GTDB-Tk v(\S+)"],
- 'dRep': ['v_dRep.txt', r"version (\S+)"]
+ 'dRep': ['v_dRep.txt', r"version (\S+)"],
+ 'tRNAscan-SE': ['v_tRNAscan.txt', r"tRNAscan-SE (\S+)"],
+ 'Barrnap': ['v_barrnap.txt', r"barrnap (\S+)"],
+ 'Prodigal': ['v_prodigal.txt', r"Prodigal (\S+):"],
}
results = OrderedDict()
results['metagWGS'] = '<span style="color:#999999;\">N/A</span>'
@@ -65,6 +67,9 @@ results['Metawrap'] = '<span style="color:#999999;\">N/A</span>'
results['dRep'] = '<span style="color:#999999;\">N/A</span>'
results['GTDBTK'] = '<span style="color:#999999;\">N/A</span>'
results['MultiQC'] = '<span style="color:#999999;\">N/A</span>'
+results['tRNAscan-SE'] = '<span style="color:#999999;\">N/A</span>'
+results['Barrnap'] = '<span style="color:#999999;\">N/A</span>'
+results['Prodigal'] = '<span style="color:#999999;\">N/A</span>'
# Search each file using its regex
for k, v in regexes.items():
--
GitLab
From 993642d2aa2bbecd8cd9568b8967bcf1fd14eb7e Mon Sep 17 00:00:00 2001
From: Jean Mainguy <jean.mainguy@inra.fr>
Date: Mon, 24 Oct 2022 15:04:34 +0200
Subject: [PATCH 059/194] clean nf scripts
---
main.nf | 5 -----
subworkflows/02_assembly.nf | 12 ++++--------
2 files changed, 4 insertions(+), 13 deletions(-)
diff --git a/main.nf b/main.nf
index 75c9f7b..87a6bb4 100644
--- a/main.nf
+++ b/main.nf
@@ -448,14 +448,9 @@ workflow {
ch_prokka_ffn = S04_STRUCTURAL_ANNOT.out.ffn
ch_prokka_faa = S04_STRUCTURAL_ANNOT.out.faa
ch_prokka_gff = S04_STRUCTURAL_ANNOT.out.gff
- // ch_prokka_fna = S04_STRUCTURAL_ANNOT.out.fna
ch_prokka_report = S04_STRUCTURAL_ANNOT.out.report
- // ch_contigs_and_reads = ch_prokka_fna
- // .join(ch_reads, remainder: true)
-
ch_software_versions_S04 = S04_STRUCTURAL_ANNOT.out.software_versions
- // exit 0, 'Exit now... S04_STRUCTURAL_ANNOT being constructed '
}
ch_bam = Channel.empty()
diff --git a/subworkflows/02_assembly.nf b/subworkflows/02_assembly.nf
index 1106df4..819c674 100644
--- a/subworkflows/02_assembly.nf
+++ b/subworkflows/02_assembly.nf
@@ -6,10 +6,10 @@ include { SAMTOOLS } from '../modules/read_alignment_metrics'
workflow STEP_02_ASSEMBLY {
take:
- reads
- assembly
- has_assembly
- assembly_tool
+ reads
+ assembly
+ has_assembly
+ assembly_tool
main:
ch_assembler_v = Channel.empty()
@@ -95,12 +95,8 @@ workflow STEP_02_ASSEMBLY {
emit:
assembly = ch_assembly_renamed
-<<<<<<< HEAD
reads = ch_reads
bam = ch_bam
-=======
- dedup = ch_dedup
->>>>>>> faec234a8f1a5d6c7e324dfc6723c758d713bd3c
idxstats = ch_idxstats
flagstat = ch_flagstat
assembly_report = ch_assembly_report
--
GitLab
From 9eb508cfd6e7ba69d2fed9d6ec36ba0bba06c5cd Mon Sep 17 00:00:00 2001
From: Jean Mainguy <jean.mainguy@inra.fr>
Date: Mon, 24 Oct 2022 15:38:20 +0200
Subject: [PATCH 060/194] remove read aln in step 05
---
main.nf | 56 +++++++++++++++++++-----------------
subworkflows/05_alignment.nf | 49 +++++++++++++++----------------
2 files changed, 53 insertions(+), 52 deletions(-)
diff --git a/main.nf b/main.nf
index 87a6bb4..5604044 100644
--- a/main.nf
+++ b/main.nf
@@ -287,7 +287,7 @@ workflow {
ch_kaiju_db = Channel.empty()
ch_eggnog_db = Channel.empty()
ch_taxonomy = Channel.empty()
- ch_diamond = Channel.empty()
+ ch_diamon_db = Channel.empty()
ch_gtbdtk_db = Channel.empty()
@@ -297,7 +297,7 @@ workflow {
ch_kaiju_db = DATABASES.out.kaiju_db
ch_eggnog_db = DATABASES.out.eggnog
ch_taxonomy = DATABASES.out.taxonomy
- ch_diamond = DATABASES.out.diamond
+ ch_diamon_db = DATABASES.out.diamond
ch_gtbdtk_db = DATABASES.out.gtdbtk
ch_multiqc_config = Channel.empty()
@@ -317,7 +317,7 @@ workflow {
ch_final_assembly_flagstat_report = Channel.empty()
ch_assembly_report = Channel.empty()
ch_filtered_report = Channel.empty()
- ch_prokka_report = Channel.empty()
+ ch_annotation_report = Channel.empty()
ch_bins_abundances_report = Channel.empty()
ch_bins_stats_report = Channel.empty()
ch_quast = Channel.empty()
@@ -409,8 +409,8 @@ workflow {
ch_reads = S02_ASSEMBLY.out.reads
ch_bam = S02_ASSEMBLY.out.bam
+ ch_sam_coverage = S02_ASSEMBLY.out.coverage
ch_idxstats = S02_ASSEMBLY.out.idxstats
-
ch_unfilter_assembly_flagstat_report = S02_ASSEMBLY.out.flagstat
ch_quast_before_filter_report = S02_ASSEMBLY.out.assembly_report
@@ -433,47 +433,51 @@ workflow {
ch_min_contigs_cpm
)
ch_assembly = S03_FILTERING.out.selected
+ ch_bam = S03_FILTERING.out.bam
+ ch_sam_coverage = S02_ASSEMBLY.out.coverage
ch_quast_after_filter_report = S03_FILTERING.out.report
-
+ ch_final_assembly_flagstat_report = S03_FILTERING.out.sam_flagstat
+
+
+
+
}
- ch_contigs_and_reads = Channel.empty()
- ch_prokka_ffn = Channel.empty()
- ch_prokka_faa = Channel.empty()
- ch_prokka_gff = Channel.empty()
- ch_prokka_fna = Channel.empty()
+ ch_annotation_ffn = Channel.empty()
+ ch_annotation_faa = Channel.empty()
+ ch_annotation_gff = Channel.empty()
if ( !params.stop_at_clean && !params.stop_at_assembly && !params.stop_at_filtering ) {
S04_STRUCTURAL_ANNOT ( ch_assembly )
- ch_prokka_ffn = S04_STRUCTURAL_ANNOT.out.ffn
- ch_prokka_faa = S04_STRUCTURAL_ANNOT.out.faa
- ch_prokka_gff = S04_STRUCTURAL_ANNOT.out.gff
- ch_prokka_report = S04_STRUCTURAL_ANNOT.out.report
+ ch_annotation_ffn = S04_STRUCTURAL_ANNOT.out.ffn
+ ch_annotation_faa = S04_STRUCTURAL_ANNOT.out.faa
+ ch_annotation_gff = S04_STRUCTURAL_ANNOT.out.gff
+ ch_annotation_report = S04_STRUCTURAL_ANNOT.out.report
ch_software_versions_S04 = S04_STRUCTURAL_ANNOT.out.software_versions
}
- ch_bam = Channel.empty()
+ // ch_bam = Channel.empty()
ch_m8 = Channel.empty()
- ch_sam_coverage = Channel.empty()
+ // ch_sam_coverage = Channel.empty()
if ( !params.stop_at_clean && !params.stop_at_assembly && !params.stop_at_filtering && !params.stop_at_structural_annot ) {
- S05_ALIGNMENT ( ch_contigs_and_reads, ch_prokka_faa, ch_diamond)
- ch_bam = S05_ALIGNMENT.out.bam
+ S05_ALIGNMENT (ch_annotation_faa, ch_diamon_db)
+ // ch_bam = S05_ALIGNMENT.out.bam
ch_m8 = S05_ALIGNMENT.out.m8
- ch_sam_coverage = S05_ALIGNMENT.out.sam_coverage
+ // ch_sam_coverage = S05_ALIGNMENT.out.sam_coverage
- if (!params.skip_filtering){
- // when filtering is skip reads vs assembly remain unchanged so no need to send it to multiqc
- ch_final_assembly_flagstat_report = S05_ALIGNMENT.out.sam_flagstat
- }
+ // if (!params.skip_filtering){
+ // // when filtering is skip reads vs assembly remain unchanged so no need to send it to multiqc
+ // // ch_final_assembly_flagstat_report = S05_ALIGNMENT.out.sam_flagstat
+ // }
ch_software_versions_S05 = S05_ALIGNMENT.out.software_versions
}
ch_quant_report = Channel.empty()
ch_v_eggnogmapper = Channel.empty()
if ( !params.stop_at_clean && !params.stop_at_assembly && !params.stop_at_filtering && !params.stop_at_structural_annot && !params.skip_func_annot ) {
- S06_FUNC_ANNOT ( ch_prokka_ffn, ch_prokka_faa, ch_prokka_gff, ch_bam, ch_m8, ch_eggnog_db )
+ S06_FUNC_ANNOT ( ch_annotation_ffn, ch_annotation_faa, ch_annotation_gff, ch_bam, ch_m8, ch_eggnog_db )
ch_quant_report = S06_FUNC_ANNOT.out.quant_report
ch_software_versions_S06 = S06_FUNC_ANNOT.out.software_versions
@@ -486,7 +490,7 @@ workflow {
}
if ( !params.stop_at_clean && !params.stop_at_assembly && !params.stop_at_filtering && !params.stop_at_structural_annot && !params.skip_binning ) {
- S08_BINNING( ch_reads, ch_prokka_fna, ch_bam, ch_gtbdtk_db, ch_quast )
+ S08_BINNING( ch_reads, ch_annotation_fna, ch_bam, ch_gtbdtk_db, ch_quast )
ch_bins_abundances_report = S08_BINNING.out.bins_abundances_report
ch_bins_stats_report = S08_BINNING.out.bins_stats_report
@@ -515,7 +519,7 @@ workflow {
ch_quast_before_filter_report.collect().ifEmpty([]),
ch_quast_after_filter_report.collect().ifEmpty([]),
ch_final_assembly_flagstat_report.collect().ifEmpty([]),
- ch_prokka_report.collect().ifEmpty([]),
+ ch_annotation_report.collect().ifEmpty([]),
ch_quant_report.collect().ifEmpty([]),
ch_bins_abundances_report.collect().ifEmpty([]),
ch_bins_stats_report.collect().ifEmpty([])
diff --git a/subworkflows/05_alignment.nf b/subworkflows/05_alignment.nf
index 1d7c543..191d20f 100644
--- a/subworkflows/05_alignment.nf
+++ b/subworkflows/05_alignment.nf
@@ -4,34 +4,34 @@ include { DIAMOND } from '../modules/diamond'
workflow STEP_05_ALIGNMENT {
take:
- contigs_and_reads
+ // contigs_and_reads
prokka_faa
diamond
main:
- ch_bwa_mem_v = Channel.empty()
- ch_minimap2_v = Channel.empty()
- ch_samtools_v = Channel.empty()
+ // ch_bwa_mem_v = Channel.empty()
+ // ch_minimap2_v = Channel.empty()
+ // ch_samtools_v = Channel.empty()
ch_diamond_v = Channel.empty()
- publishDir = "05_alignment/05_1_reads_alignment_on_contigs"
- if (params.type == 'SR') {
- BWA_MEM(contigs_and_reads, publishDir)
- ch_bam = BWA_MEM.out.bam
+ // publishDir = "05_alignment/05_1_reads_alignment_on_contigs"
+ // if (params.type == 'SR') {
+ // BWA_MEM(contigs_and_reads, publishDir)
+ // ch_bam = BWA_MEM.out.bam
- ch_bwa_mem_v = BWA_MEM.out.v_bwa_mem2
- ch_samtools_v = BWA_MEM.out.v_samtools
- } else {
- MINIMAP2(contigs_and_reads, publishDir)
- ch_bam = MINIMAP2.out.bam
+ // ch_bwa_mem_v = BWA_MEM.out.v_bwa_mem2
+ // ch_samtools_v = BWA_MEM.out.v_samtools
+ // } else {
+ // MINIMAP2(contigs_and_reads, publishDir)
+ // ch_bam = MINIMAP2.out.bam
- ch_minimap2_v = MINIMAP2.out.v_minimap2
- ch_samtools_v = MINIMAP2.out.v_samtools
- }
+ // ch_minimap2_v = MINIMAP2.out.v_minimap2
+ // ch_samtools_v = MINIMAP2.out.v_samtools
+ // }
- SAMTOOLS(ch_bam, publishDir)
- ch_sam_coverage = SAMTOOLS.out.sam_coverage
- ch_sam_flagstat = SAMTOOLS.out.sam_flagstat
+ // SAMTOOLS(ch_bam, publishDir)
+ // ch_sam_coverage = SAMTOOLS.out.sam_coverage
+ // ch_sam_flagstat = SAMTOOLS.out.sam_flagstat
ch_m8 =Channel.empty()
if (!params.skip_func_annot || !params.skip_taxo_affi){
@@ -42,13 +42,10 @@ workflow STEP_05_ALIGNMENT {
ch_m8 = DIAMOND.out.m8
ch_diamond_v = DIAMOND.out.v_diamond
}
- ch_software_versions = ch_bwa_mem_v.first().mix(ch_minimap2_v.first(),
- ch_samtools_v.first(),
- ch_diamond_v.first())
emit:
- bam = ch_bam
+ // bam = ch_bam
m8 = ch_m8
- sam_coverage = ch_sam_coverage
- software_versions = ch_software_versions
- sam_flagstat = ch_sam_flagstat
+ // sam_coverage = ch_sam_coverage
+ software_versions = ch_diamond_v.first()
+ // sam_flagstat = ch_sam_flagstat
}
--
GitLab
From 86f50821dec927f08e3829563e6bf0e4a3168ee1 Mon Sep 17 00:00:00 2001
From: Jean Mainguy <jean.mainguy@inra.fr>
Date: Mon, 24 Oct 2022 15:39:33 +0200
Subject: [PATCH 061/194] reorganise 02_assembly.nf
---
modules/read_alignment_metrics.nf | 4 +++-
subworkflows/02_assembly.nf | 20 +++++++++-----------
2 files changed, 12 insertions(+), 12 deletions(-)
diff --git a/modules/read_alignment_metrics.nf b/modules/read_alignment_metrics.nf
index e23dc78..ee1bd54 100644
--- a/modules/read_alignment_metrics.nf
+++ b/modules/read_alignment_metrics.nf
@@ -11,12 +11,14 @@ process SAMTOOLS {
tuple val(meta), path("${meta.id}.idxstats"), emit: sam_idxstat
path "${meta.id}.flagstat", emit: sam_flagstat
path "${meta.id}*"
-
+ path "v_samtools.txt", emit: v_samtools
script:
"""
samtools flagstat -@ ${task.cpus} ${bam} > ${meta.id}.flagstat
samtools coverage ${bam} > ${meta.id}.coverage.tsv
samtools idxstats ${bam} > ${meta.id}.idxstats
+
+ samtools --version &> v_samtools.txt
"""
}
\ No newline at end of file
diff --git a/subworkflows/02_assembly.nf b/subworkflows/02_assembly.nf
index 819c674..ee5a207 100644
--- a/subworkflows/02_assembly.nf
+++ b/subworkflows/02_assembly.nf
@@ -64,12 +64,7 @@ workflow STEP_02_ASSEMBLY {
ch_reads = READS_DEDUPLICATION.out.dedup_reads
ch_bam = READS_DEDUPLICATION.out.bam
- SAMTOOLS(ch_bam, "02_assembly")
- ch_idxstats = SAMTOOLS.out.sam_idxstat
- ch_flagstat = SAMTOOLS.out.sam_flagstat
-
ch_bwa_mem_v = READS_DEDUPLICATION.out.v_bwa_mem2
- ch_samtools_v = READS_DEDUPLICATION.out.v_samtools
} else {
@@ -77,16 +72,18 @@ workflow STEP_02_ASSEMBLY {
MINIMAP2(ch_contigs_and_reads,"02_assembly")
ch_bam = MINIMAP2.out.bam
- SAMTOOLS(ch_bam, "02_assembly")
- ch_idxstats = SAMTOOLS.out.sam_idxstat
- ch_flagstat = SAMTOOLS.out.sam_flagstat
-
ch_minimap2_v = MINIMAP2.out.v_minimap2
- ch_samtools_v = MINIMAP2.out.v_samtools
+
}
-
}
+ SAMTOOLS(ch_bam, "02_assembly")
+
+ ch_idxstats = SAMTOOLS.out.sam_idxstat
+ ch_flagstat = SAMTOOLS.out.sam_flagstat
+ ch_coverage = SAMTOOLS.out.sam_coverage
+ ch_samtools_v = SAMTOOLS.out.v_samtools
+
ch_software_versions = ch_assembler_v.first().mix( ch_quast_v.first(),
ch_bwa_mem_v.first(),
ch_minimap2_v.first(),
@@ -99,6 +96,7 @@ workflow STEP_02_ASSEMBLY {
bam = ch_bam
idxstats = ch_idxstats
flagstat = ch_flagstat
+ coverage = ch_coverage
assembly_report = ch_assembly_report
software_versions = ch_software_versions
}
\ No newline at end of file
--
GitLab
From bdebbf18cb3f6e8e326d2d5e3e6f91d275b25672 Mon Sep 17 00:00:00 2001
From: Jean Mainguy <jean.mainguy@inra.fr>
Date: Mon, 24 Oct 2022 15:59:42 +0200
Subject: [PATCH 062/194] rename diamond result channel for better clarity
---
main.nf | 10 +++++-----
1 file changed, 5 insertions(+), 5 deletions(-)
diff --git a/main.nf b/main.nf
index 5604044..173bf84 100644
--- a/main.nf
+++ b/main.nf
@@ -434,7 +434,7 @@ workflow {
)
ch_assembly = S03_FILTERING.out.selected
ch_bam = S03_FILTERING.out.bam
- ch_sam_coverage = S02_ASSEMBLY.out.coverage
+ ch_sam_coverage = S03_FILTERING.out.coverage
ch_quast_after_filter_report = S03_FILTERING.out.report
ch_final_assembly_flagstat_report = S03_FILTERING.out.sam_flagstat
@@ -458,13 +458,13 @@ workflow {
}
// ch_bam = Channel.empty()
- ch_m8 = Channel.empty()
+ ch_diamond_result = Channel.empty()
// ch_sam_coverage = Channel.empty()
if ( !params.stop_at_clean && !params.stop_at_assembly && !params.stop_at_filtering && !params.stop_at_structural_annot ) {
S05_ALIGNMENT (ch_annotation_faa, ch_diamon_db)
// ch_bam = S05_ALIGNMENT.out.bam
- ch_m8 = S05_ALIGNMENT.out.m8
+ ch_diamond_result = S05_ALIGNMENT.out.m8
// ch_sam_coverage = S05_ALIGNMENT.out.sam_coverage
// if (!params.skip_filtering){
@@ -477,14 +477,14 @@ workflow {
ch_quant_report = Channel.empty()
ch_v_eggnogmapper = Channel.empty()
if ( !params.stop_at_clean && !params.stop_at_assembly && !params.stop_at_filtering && !params.stop_at_structural_annot && !params.skip_func_annot ) {
- S06_FUNC_ANNOT ( ch_annotation_ffn, ch_annotation_faa, ch_annotation_gff, ch_bam, ch_m8, ch_eggnog_db )
+ S06_FUNC_ANNOT ( ch_annotation_ffn, ch_annotation_faa, ch_annotation_gff, ch_bam, ch_diamond_result, ch_eggnog_db )
ch_quant_report = S06_FUNC_ANNOT.out.quant_report
ch_software_versions_S06 = S06_FUNC_ANNOT.out.software_versions
}
if ( !params.stop_at_clean && !params.stop_at_assembly && !params.stop_at_filtering && !params.stop_at_structural_annot && !params.skip_taxo_affi ) {
- S07_TAXO_AFFI ( ch_taxonomy, ch_m8, ch_sam_coverage)
+ S07_TAXO_AFFI ( ch_taxonomy, ch_diamond_result, ch_sam_coverage)
ch_software_versions_S07 = S07_TAXO_AFFI.out.software_versions
}
--
GitLab
From 0cc6fa08c68ca56a56b9baa91c67eb7095314a4d Mon Sep 17 00:00:00 2001
From: Jean Mainguy <jean.mainguy@inra.fr>
Date: Mon, 24 Oct 2022 16:00:02 +0200
Subject: [PATCH 063/194] rm graph as expected output
---
modules/assign_taxonomy.nf | 1 -
1 file changed, 1 deletion(-)
diff --git a/modules/assign_taxonomy.nf b/modules/assign_taxonomy.nf
index 5b03ec4..559c06c 100644
--- a/modules/assign_taxonomy.nf
+++ b/modules/assign_taxonomy.nf
@@ -11,7 +11,6 @@ process ASSIGN_TAXONOMY {
tuple val(meta.id), path("${meta.id}.percontig.tsv"), emit: t_percontig
tuple val(meta.id), path("${meta.id}.pergene.tsv"), emit: t_pergene
tuple val(meta.id), path("${meta.id}.warn.tsv"), emit: t_warn
- tuple val(meta.id), path("graphs"), emit: t_graphs
path "${meta.id}_quantif_percontig.tsv", emit: q_all
path "${meta.id}_quantif_percontig_by_superkingdom.tsv", emit: q_superkingdom
path "${meta.id}_quantif_percontig_by_phylum.tsv", emit: q_phylum
--
GitLab
From 3a3302317ed10342b431b6b83f9bfc1d53690f92 Mon Sep 17 00:00:00 2001
From: Jean Mainguy <jean.mainguy@inra.fr>
Date: Mon, 24 Oct 2022 16:53:27 +0200
Subject: [PATCH 064/194] refactoring of filter_contig_per_cpm.py using pyfastx
#203
---
bin/Filter_contig_per_cpm.py | 95 --------------------------------
bin/filter_contig_per_cpm.py | 103 +++++++++++++++++++++++++++++++++++
conf/base.config | 4 +-
conf/test.config | 2 +-
main.nf | 23 +++-----
modules/filtering_cpm.nf | 2 +-
subworkflows/03_filtering.nf | 20 +++++--
7 files changed, 131 insertions(+), 118 deletions(-)
delete mode 100755 bin/Filter_contig_per_cpm.py
create mode 100755 bin/filter_contig_per_cpm.py
diff --git a/bin/Filter_contig_per_cpm.py b/bin/Filter_contig_per_cpm.py
deleted file mode 100755
index 9127e57..0000000
--- a/bin/Filter_contig_per_cpm.py
+++ /dev/null
@@ -1,95 +0,0 @@
-#!/usr/bin/env python
-
-"""----------------------------------------------------------------------------
- Script Name: Filter_contig_per_cpm.py
- Description: Calculates the CPM normalization of mapped reads for each \
- contig and returns contigs which have a CPM > cutoff in .fa.
- Input files: Samtools idxstats output file, .fasta file of contigs.
- Created By: Joanna Fourquet
- Date: 2020-04-01
--------------------------------------------------------------------------------
-"""
-
-# Metadata
-__author__ = 'Joanna Fourquet \
-- GenPhySE - Team NED'
-__copyright__ = 'Copyright (C) 2020 INRA'
-__license__ = 'GNU General Public License'
-__version__ = '0.1'
-__email__ = 'support.bioinfo.genotoul@inra.fr'
-__status__ = 'dev'
-
-# Status: dev
-
-# Modules importation
-
-try:
- import argparse
- import sys
- import pandas as p
- import numpy as np
- from Bio.Seq import Seq
- from Bio.SeqRecord import SeqRecord
- import pprint
- from Bio import SeqIO
-except ImportError as error:
- print(error)
- exit(1)
-
-################################################
-# Function
-################################################
-
-def cpm(counts):
- N = np.sum(counts.iloc[:,2], axis=0)
- C = counts.iloc[:,2]
- cpm_values = 1e6 * C / N
- return(cpm_values)
-
-def main(argv):
- parser = argparse.ArgumentParser()
- parser.add_argument("-i", "--samtools_idxstats", \
- required = True, help = "samtools idxstats file containing contig id, \
- sequence length, number of mapped reads or fragments, \
- number of unmapped reads or fragments")
- parser.add_argument('-f', '--fasta_file', required = True, help = \
- 'fasta file containing sequences of contigs.')
- parser.add_argument("-c", "--cutoff_cpm", required = True, \
- help = "Minimum number of reads in a contig")
- parser.add_argument("-s", "--select", \
- help = "Name of outpout .fa file containing contigs which passed cpm cutoff")
- parser.add_argument("-d", "--discard", \
- help = "Name of outpout .fa file containing contigs which don't passed cpm cutoff")
- args = parser.parse_args()
-
- # Read input table
- raw_counts = p.read_table(args.samtools_idxstats,sep ='\t',header = None, comment="*")
-
- # Calculates cpm for each contig
- res_cpm = cpm(raw_counts)
-
- cutoff = float(args.cutoff_cpm)
- # Contigs with nb reads > cutoff
- kept_contigs = raw_counts.iloc[np.where(res_cpm >= cutoff)[0],0]
-
- # Contigs with nb reads < cutoff
- unkept_contigs = raw_counts.iloc[np.where(res_cpm < cutoff)[0],0]
-
- # Write new fasta files with kept and unkept contigs
- with open(args.fasta_file, "rU") as fasta_file,\
- open(args.select, "w") as out_select_handle,\
- open(args.discard, "w") as out_discard_handle:
- for record in SeqIO.parse(fasta_file, "fasta"):
- try :
- contig_id = record.id
- if(contig_id in list(kept_contigs)):
- SeqIO.write(record, out_select_handle, "fasta")
- else:
- if(contig_id in list(unkept_contigs)):
- SeqIO.write(record, out_discard_handle, "fasta")
- except :
- print ("Warning input fasta file: contig " + record.id + " issue")
- pass
-
-if __name__ == "__main__":
- main(sys.argv[1:])
diff --git a/bin/filter_contig_per_cpm.py b/bin/filter_contig_per_cpm.py
new file mode 100755
index 0000000..ba563ec
--- /dev/null
+++ b/bin/filter_contig_per_cpm.py
@@ -0,0 +1,103 @@
+#!/usr/bin/env python
+
+"""----------------------------------------------------------------------------
+ Script Name: Filter_contig_per_cpm.py
+ Description: Calculates the CPM normalization of mapped reads for each \
+ contig and returns contigs which have a CPM > cutoff in .fa.
+ Input files: Samtools idxstats output file, .fasta file of contigs.
+ Created By: Joanna Fourquet
+ Date: 2020-04-01
+-------------------------------------------------------------------------------
+"""
+
+# Metadata
+__author__ = 'Joanna Fourquet \
+- GenPhySE - Team NED'
+__copyright__ = 'Copyright (C) 2020 INRA'
+__license__ = 'GNU General Public License'
+__version__ = '0.1'
+__email__ = 'support.bioinfo.genotoul@inra.fr'
+__status__ = 'dev'
+
+# Status: dev
+
+# Modules importation
+
+
+from argparse import ArgumentParser, ArgumentDefaultsHelpFormatter
+import sys
+import pandas as pd
+import numpy as np
+import logging
+from Bio import SeqIO
+import pyfastx
+
+################################################
+# Function
+################################################
+
+def parse_arguments():
+ """Parse script arguments."""
+ parser = ArgumentParser(description="...",
+ formatter_class=ArgumentDefaultsHelpFormatter)
+
+ parser.add_argument("-i", "--samtools_idxstats", required = True,
+ help = "samtools idxstats file containing contig id, \
+ sequence length, number of mapped reads or fragments, \
+ number of unmapped reads or fragments")
+
+ parser.add_argument('-f', '--fasta_file', required = True,
+ help = 'fasta file containing sequences of contigs.')
+ parser.add_argument("-c", "--cutoff_cpm", required = True,
+ help = "Minimum number of reads in a contig")
+ parser.add_argument("-s", "--select",
+ help = "Name of outpout .fa file containing contigs which passed cpm cutoff")
+ parser.add_argument("-d", "--discard",
+ help = "Name of outpout .fa file containing contigs which don't passed cpm cutoff")
+
+
+ parser.add_argument("-v", "--verbose", help="increase output verbosity",
+ action="store_true")
+
+ args = parser.parse_args()
+ return args
+
+def cpm(counts):
+ N = np.sum(counts.iloc[:,2], axis=0)
+ C = counts.iloc[:,2]
+ cpm_values = 1e6 * C / N
+ return(cpm_values)
+
+def main(argv):
+ args = parse_arguments()
+
+ if args.verbose:
+ logging.basicConfig(format="%(levelname)s: %(message)s", level=logging.DEBUG)
+ logging.info('Mode verbose ON')
+
+ else:
+ logging.basicConfig(format="%(levelname)s: %(message)s")
+
+
+ # Read input table
+ raw_counts = pd.read_table(args.samtools_idxstats,sep ='\t',header = None, comment="*")
+
+ # Calculates cpm for each contig
+ res_cpm = cpm(raw_counts)
+
+ cutoff = float(args.cutoff_cpm)
+ # Contigs with nb reads > cutoff
+ kept_contigs = raw_counts.iloc[np.where(res_cpm >= cutoff)[0],0]
+
+
+ # Write new fasta files with kept and unkept contigs
+ with open(args.select, "w") as out_select_handle, open(args.discard, "w") as out_discard_handle:
+
+ for contig, seq in pyfastx.Fasta(args.fasta_file, build_index=False):
+ if contig in kept_contigs:
+ out_select_handle.write(f'>{contig}\n{seq}\n')
+ else:
+ out_discard_handle.write(f'>{contig}\n{seq}\n')
+
+if __name__ == "__main__":
+ main(sys.argv[1:])
diff --git a/conf/base.config b/conf/base.config
index a94490b..c5de8a3 100644
--- a/conf/base.config
+++ b/conf/base.config
@@ -55,8 +55,8 @@ process {
memory = { 32.GB * task.attempt }
}
withLabel: ASSEMBLY_FILTER {
- memory = { 8.GB * task.attempt }
- cpus = 4
+ memory = { 2.GB * task.attempt }
+ cpus = 1
}
withLabel: CD_HIT {
memory = { 50.GB * task.attempt }
diff --git a/conf/test.config b/conf/test.config
index ae50b57..8b437cd 100644
--- a/conf/test.config
+++ b/conf/test.config
@@ -47,7 +47,7 @@ process {
}
withLabel: ASSEMBLY_FILTER {
memory = { 1.GB * task.attempt }
- cpus = 2
+ cpus = 1
}
withLabel: CD_HIT {
memory = { 16.GB * task.attempt }
diff --git a/main.nf b/main.nf
index 173bf84..2c53770 100644
--- a/main.nf
+++ b/main.nf
@@ -418,28 +418,23 @@ workflow {
}
if ( !params.stop_at_clean && !params.stop_at_assembly && !params.skip_filtering ) {
+ print("IN FILTERING")
ch_min_contigs_cpm = Channel.value(params.min_contigs_cpm)
- ch_assembly
- .splitFasta(by: 100000, file: true)
- .set{ch_chunk_assembly_for_filter}
- ch_chunk_assembly_for_filter
- .combine(ch_idxstats, by:0)
- .set{ch_assembly_and_idxstats}
S03_FILTERING (
- ch_assembly_and_idxstats,
+ ch_assembly,
+ ch_idxstats,
+ ch_bam,
ch_min_contigs_cpm
)
- ch_assembly = S03_FILTERING.out.selected
+
+ ch_assembly = S03_FILTERING.out.selected_contigs
ch_bam = S03_FILTERING.out.bam
- ch_sam_coverage = S03_FILTERING.out.coverage
- ch_quast_after_filter_report = S03_FILTERING.out.report
- ch_final_assembly_flagstat_report = S03_FILTERING.out.sam_flagstat
-
-
-
+ // ch_sam_coverage = S03_FILTERING.out.coverage
+ ch_quast_after_filter_report = S03_FILTERING.out.quast_report
+ // ch_final_assembly_flagstat_report = S03_FILTERING.out.sam_flagstat
}
diff --git a/modules/filtering_cpm.nf b/modules/filtering_cpm.nf
index 951a201..0155034 100644
--- a/modules/filtering_cpm.nf
+++ b/modules/filtering_cpm.nf
@@ -12,7 +12,7 @@ process CHUNK_ASSEMBLY_FILTER {
script:
chunk_name = assembly_file.baseName
"""
- Filter_contig_per_cpm.py -i ${idxstats} -f ${assembly_file} -c ${min_cpm} -s ${chunk_name}_select_cpm${min_cpm}.fasta -d ${chunk_name}_discard_cpm${min_cpm}.fasta
+ filter_contig_per_cpm.py -i ${idxstats} -f ${assembly_file} -c ${min_cpm} -s ${chunk_name}_select_cpm${min_cpm}.fasta -d ${chunk_name}_discard_cpm${min_cpm}.fasta
"""
}
diff --git a/subworkflows/03_filtering.nf b/subworkflows/03_filtering.nf
index b13e490..488a576 100644
--- a/subworkflows/03_filtering.nf
+++ b/subworkflows/03_filtering.nf
@@ -3,12 +3,21 @@ include { QUAST as FILTERED_QUAST } from '../modules/metaquast'
workflow STEP_03_ASSEMBLY_FILTER {
take:
- assembly_and_idxstats
+ assemblies
+ idxstats
+ bam
min_cpm
main:
+ ch_chunk_assembly_for_filter = assemblies
+ .splitFasta(by: 100000, file: true)
+
+
+ ch_assembly_and_idxstats = ch_chunk_assembly_for_filter
+ .combine(idxstats, by:0)
+
CHUNK_ASSEMBLY_FILTER (
- assembly_and_idxstats,
+ ch_assembly_and_idxstats,
min_cpm
)
ch_chunk_selected = CHUNK_ASSEMBLY_FILTER.out.chunk_selected
@@ -30,10 +39,11 @@ workflow STEP_03_ASSEMBLY_FILTER {
ch_merged_selected = MERGE_ASSEMBLY_FILTER.out.merged_selected
FILTERED_QUAST( ch_merged_selected, "03_filtering/quast_filtered" )
- ch_filtered_report = FILTERED_QUAST.out.report
+ ch_quast_report = FILTERED_QUAST.out.report
emit:
- selected = ch_merged_selected
- report = ch_filtered_report
+ selected_contigs = ch_merged_selected
+ quast_report = ch_quast_report
+ bam = bam
}
\ No newline at end of file
--
GitLab
From 60296922bd85c849aaacab08b48218934db8af9f Mon Sep 17 00:00:00 2001
From: Jean Mainguy <jean.mainguy@inra.fr>
Date: Mon, 24 Oct 2022 18:20:34 +0200
Subject: [PATCH 065/194] compelete refactoring of filter_contig_per_cpm.py
---
bin/filter_contig_per_cpm.py | 42 +++++++++++++++++++-----------------
1 file changed, 22 insertions(+), 20 deletions(-)
diff --git a/bin/filter_contig_per_cpm.py b/bin/filter_contig_per_cpm.py
index ba563ec..8f01cec 100755
--- a/bin/filter_contig_per_cpm.py
+++ b/bin/filter_contig_per_cpm.py
@@ -2,18 +2,17 @@
"""----------------------------------------------------------------------------
Script Name: Filter_contig_per_cpm.py
- Description: Calculates the CPM normalization of mapped reads for each \
+ Description: Calculates the CPM normalization of mapped reads for each
contig and returns contigs which have a CPM > cutoff in .fa.
Input files: Samtools idxstats output file, .fasta file of contigs.
- Created By: Joanna Fourquet
- Date: 2020-04-01
+ Created By: Jean Mainguy
+ Date: 2022-24-10
-------------------------------------------------------------------------------
"""
# Metadata
-__author__ = 'Joanna Fourquet \
-- GenPhySE - Team NED'
-__copyright__ = 'Copyright (C) 2020 INRA'
+__author__ = 'Mainguy Jean - Plateforme bioinformatique Toulouse'
+__copyright__ = 'Copyright (C) 2022 INRAE'
__license__ = 'GNU General Public License'
__version__ = '0.1'
__email__ = 'support.bioinfo.genotoul@inra.fr'
@@ -25,11 +24,9 @@ __status__ = 'dev'
from argparse import ArgumentParser, ArgumentDefaultsHelpFormatter
-import sys
import pandas as pd
import numpy as np
import logging
-from Bio import SeqIO
import pyfastx
################################################
@@ -62,13 +59,8 @@ def parse_arguments():
args = parser.parse_args()
return args
-def cpm(counts):
- N = np.sum(counts.iloc[:,2], axis=0)
- C = counts.iloc[:,2]
- cpm_values = 1e6 * C / N
- return(cpm_values)
-def main(argv):
+def main():
args = parse_arguments()
if args.verbose:
@@ -78,18 +70,28 @@ def main(argv):
else:
logging.basicConfig(format="%(levelname)s: %(message)s")
+ cpm_cutoff = float(args.cutoff_cpm)
# Read input table
- raw_counts = pd.read_table(args.samtools_idxstats,sep ='\t',header = None, comment="*")
+ idxstat_df = pd.read_csv(args.samtools_idxstats,
+ sep ='\t',
+ names = ['reference_sequence_name',
+ 'sequence_length',
+ '# mapped_read-segments',
+ '#_unmapped_read-segments'],
+ comment="*")
# Calculates cpm for each contig
- res_cpm = cpm(raw_counts)
+ sum_reads = idxstat_df['# mapped_read-segments'].sum()
- cutoff = float(args.cutoff_cpm)
- # Contigs with nb reads > cutoff
- kept_contigs = raw_counts.iloc[np.where(res_cpm >= cutoff)[0],0]
+ logging.info(f'With a cpm cutoff of {args.cutoff_cpm}, contigs with less than {(sum_reads*cpm_cutoff)/1e6} reads are removed.')
+ idxstat_df['cpm_count'] = 1e6 * idxstat_df['# mapped_read-segments']/sum_reads
+
+ # Contigs with nb reads > cutoff
+ kept_contigs = idxstat_df.loc[idxstat_df["cpm_count"] >= cpm_cutoff, "reference_sequence_name"].to_list()
+ logging.info(f'{len(kept_contigs)}/{len(idxstat_df)} contigs are kept with a cpm cutoff of {cpm_cutoff}.')
# Write new fasta files with kept and unkept contigs
with open(args.select, "w") as out_select_handle, open(args.discard, "w") as out_discard_handle:
@@ -100,4 +102,4 @@ def main(argv):
out_discard_handle.write(f'>{contig}\n{seq}\n')
if __name__ == "__main__":
- main(sys.argv[1:])
+ main()
--
GitLab
From cdd78ec8ec3809914c294356cd1213fcac9aaff3 Mon Sep 17 00:00:00 2001
From: Jean Mainguy <jean.mainguy@inra.fr>
Date: Mon, 24 Oct 2022 19:00:10 +0200
Subject: [PATCH 066/194] add possibility to process multiple idxstat in
filter_contig_per_cpm.py
---
bin/filter_contig_per_cpm.py | 53 ++++++++++++++++++++++++++----------
1 file changed, 39 insertions(+), 14 deletions(-)
diff --git a/bin/filter_contig_per_cpm.py b/bin/filter_contig_per_cpm.py
index 8f01cec..fdea68d 100755
--- a/bin/filter_contig_per_cpm.py
+++ b/bin/filter_contig_per_cpm.py
@@ -38,16 +38,16 @@ def parse_arguments():
parser = ArgumentParser(description="...",
formatter_class=ArgumentDefaultsHelpFormatter)
- parser.add_argument("-i", "--samtools_idxstats", required = True,
+ parser.add_argument("-i", "--samtools_idxstats", nargs='+', required = True,
help = "samtools idxstats file containing contig id, \
sequence length, number of mapped reads or fragments, \
number of unmapped reads or fragments")
- parser.add_argument('-f', '--fasta_file', required = True,
+ parser.add_argument('-f', '--fasta_file', required = True,
help = 'fasta file containing sequences of contigs.')
parser.add_argument("-c", "--cutoff_cpm", required = True,
help = "Minimum number of reads in a contig")
- parser.add_argument("-s", "--select",
+ parser.add_argument("-s", "--select",
help = "Name of outpout .fa file containing contigs which passed cpm cutoff")
parser.add_argument("-d", "--discard",
help = "Name of outpout .fa file containing contigs which don't passed cpm cutoff")
@@ -59,6 +59,36 @@ def parse_arguments():
args = parser.parse_args()
return args
+def combine_idxstat_files(idxstat_files):
+ """
+ Combine multiple idxstat files that have the same contigs.
+
+ Sum the #_mapped_read_segments column over multiple idxstat files that have the same reference sequences.
+ """
+ columns_names = ['reference_sequence_name',
+ 'sequence_length',
+ '#_mapped_read_segments',
+ '#_unmapped_read-segments']
+
+ idxstat_df = pd.read_csv(idxstat_files[0],
+ sep ='\t',
+ names = columns_names,
+ usecols = ['reference_sequence_name',
+ 'sequence_length',
+ '#_mapped_read_segments',],
+ comment="*").set_index('reference_sequence_name')
+
+ for idxstat_file in idxstat_files[1:]:
+ other_idxstat_df = pd.read_csv(idxstat_file,
+ sep ='\t',
+ names = columns_names,
+ usecols = ['reference_sequence_name',
+ '#_mapped_read_segments',],
+ comment="*").set_index('reference_sequence_name')
+
+ idxstat_df['#_mapped_read_segments'] += other_idxstat_df['#_mapped_read_segments']
+
+ return idxstat_df
def main():
args = parse_arguments()
@@ -72,24 +102,19 @@ def main():
cpm_cutoff = float(args.cutoff_cpm)
- # Read input table
- idxstat_df = pd.read_csv(args.samtools_idxstats,
- sep ='\t',
- names = ['reference_sequence_name',
- 'sequence_length',
- '# mapped_read-segments',
- '#_unmapped_read-segments'],
- comment="*")
+ # Read input tables
+ idxstat_df = combine_idxstat_files(args.samtools_idxstats)
# Calculates cpm for each contig
- sum_reads = idxstat_df['# mapped_read-segments'].sum()
+ sum_reads = idxstat_df['#_mapped_read_segments'].sum()
+ logging.info(f'Total number of mapped reads {sum_reads}')
logging.info(f'With a cpm cutoff of {args.cutoff_cpm}, contigs with less than {(sum_reads*cpm_cutoff)/1e6} reads are removed.')
- idxstat_df['cpm_count'] = 1e6 * idxstat_df['# mapped_read-segments']/sum_reads
+ idxstat_df['cpm_count'] = 1e6 * idxstat_df['#_mapped_read_segments']/sum_reads
# Contigs with nb reads > cutoff
- kept_contigs = idxstat_df.loc[idxstat_df["cpm_count"] >= cpm_cutoff, "reference_sequence_name"].to_list()
+ kept_contigs = idxstat_df.loc[idxstat_df["cpm_count"] >= cpm_cutoff].index
logging.info(f'{len(kept_contigs)}/{len(idxstat_df)} contigs are kept with a cpm cutoff of {cpm_cutoff}.')
# Write new fasta files with kept and unkept contigs
--
GitLab
From aebc8ad4d19434aedf1d864f6c54e83087c3c2ac Mon Sep 17 00:00:00 2001
From: Jean Mainguy <jean.mainguy@inra.fr>
Date: Tue, 25 Oct 2022 12:01:03 +0200
Subject: [PATCH 067/194] add structure and process to rescue reads in
discarded contigs
---
subworkflows/03_filtering.nf | 125 +++++++++++++++++++++++++++++++++--
1 file changed, 120 insertions(+), 5 deletions(-)
diff --git a/subworkflows/03_filtering.nf b/subworkflows/03_filtering.nf
index 488a576..dab2324 100644
--- a/subworkflows/03_filtering.nf
+++ b/subworkflows/03_filtering.nf
@@ -1,5 +1,7 @@
include { CHUNK_ASSEMBLY_FILTER; MERGE_ASSEMBLY_FILTER} from '../modules/filtering_cpm.nf'
-include { QUAST as FILTERED_QUAST } from '../modules/metaquast'
+include { QUAST } from '../modules/metaquast'
+include { MINIMAP2; BWA_MEM } from '../modules/read_alignment'
+include { SAMTOOLS } from '../modules/read_alignment_metrics'
workflow STEP_03_ASSEMBLY_FILTER {
take:
@@ -37,13 +39,126 @@ workflow STEP_03_ASSEMBLY_FILTER {
min_cpm
)
ch_merged_selected = MERGE_ASSEMBLY_FILTER.out.merged_selected
+ discarded_contigs = MERGE_ASSEMBLY_FILTER.out.merged_discarded
- FILTERED_QUAST( ch_merged_selected, "03_filtering/quast_filtered" )
- ch_quast_report = FILTERED_QUAST.out.report
+ discarded_contigs_category = discarded_contigs.branch{
+ without: it[1].isEmpty()
+ with: !it[1].isEmpty()
+ }
+ // discarded_contigs_category.view()
+ samples_with_discarded_contigs = discarded_contigs_category.with
+ samples_without_discarded_contigs = discarded_contigs_category.without
+
+ discarded_contigs_category.without.view { "WITHOUT::::::::: > $it" }
+ discarded_contigs_category.with.view { "WITH::::::::: > $it" }
+
+ ch_discarded_contigs_and_bam = samples_with_discarded_contigs.join(bam)
+ ch_discarded_contigs_and_bam.view()
+
+ FILTER_BAM(ch_discarded_contigs_and_bam)
+
+ ch_discarded_bam = FILTER_BAM.out.discarded_contigs_bam
+ ch_selected_bam = FILTER_BAM.out.selected_contigs_bam
+
+ if ( params.type.toUpperCase() == "SR" ) {
+
+ ch_discarded_reads = EXTRACT_PAIRED_READS(ch_discarded_bam)
+
+ ch_contigs_and_discarded_reads = ch_merged_selected.join(ch_discarded_reads)
+
+ BWA_MEM(ch_contigs_and_discarded_reads, "03_filtering")
+ ch_bam_with_discarded_reads = BWA_MEM.out.bam
+
+ } else {
+ ch_discarded_reads = EXTRACT_SINGLE_READS(ch_discarded_bam)
+ ch_contigs_and_discarded_reads = ch_merged_selected.join(ch_discarded_reads)
+ MINIMAP2(ch_contigs_and_reads, "03_filtering")
+ ch_bam_with_discarded_reads = MINIMAP2.out.bam
+
+ }
+
+
+ // ch_merged_bam = MERGE_BAM_FILES(ch_filtered_bam, ch_bam_with_discarded_reads)
+ // SAMTOOLS(ch_merged_bam, '03_filtering/')
+
+
+ ch_merged_bam = Channel.empty()
+ ch_coverage = Channel.empty()
+
+ ch_flagstat= Channel.empty()
+
+ // ch_flagstat = SAMTOOLS.out.sam_flagstat
+ // ch_coverage = SAMTOOLS.out.sam_coverage
+
+ QUAST( ch_merged_selected, "03_filtering/quast_filtered" )
+ ch_quast_report = QUAST.out.report
emit:
selected_contigs = ch_merged_selected
quast_report = ch_quast_report
- bam = bam
-}
\ No newline at end of file
+ bam = ch_merged_bam
+ sam_coverage = ch_coverage
+ sam_flagstat = ch_flagstat
+
+}
+
+
+process FILTER_BAM {
+ tag "${meta.id}"
+
+ input:
+ tuple val(meta), path(discarded_contigs), path(bam), path(bai)
+
+ output:
+ tuple val(meta), path("selected_contigs.bam"), emit: selected_contigs_bam
+ tuple val(meta), path("discarded_contigs.bam"), emit: discarded_contigs_bam
+
+
+ script:
+ """
+
+ samtools faidx $discarded_contigs
+ awk 'BEGIN {FS="\\t"}; {print \$1 FS "0" FS \$2}' ${discarded_contigs}.fai > discarded_contigs.bed
+
+
+ samtools view -@ ${task.cpus} -bL discarded_contigs.bed -U selected_contigs.bam $bam -o discarded_contigs.bam
+
+
+ """
+}
+
+process EXTRACT_PAIRED_READS {
+ tag "${meta.id}"
+
+ input:
+ tuple val(meta), path(bam) //, path(bai)
+
+ output:
+ tuple val(meta), path("reads*.fastq.gz"), emit: reads
+
+
+ script:
+ """
+
+ samtools fastq -@ ${task.cpus} -N -1 reads_R1.fastq.gz -2 reads_R2.fastq.gz $bam
+
+ """
+}
+
+process EXTRACT_SINGLE_READS {
+ tag "${meta.id}"
+
+ input:
+ tuple val(meta), path(bam), path(bai)
+
+ output:
+ tuple val(meta), path("reads.fastq.gz"), emit: reads
+
+ script:
+ """
+
+ samtools fastq -@ ${task.cpus} -N -1 reads.fastq.gz $bam
+
+ """
+}
--
GitLab
From d7cc93b0cb63bfce85f2d95077cf04bf8b8a9b9d Mon Sep 17 00:00:00 2001
From: Jean Mainguy <jean.mainguy@inra.fr>
Date: Tue, 25 Oct 2022 16:28:37 +0200
Subject: [PATCH 068/194] finish rescuing reads mapping to discareded contigs
---
subworkflows/03_filtering.nf | 54 +++++++++++++++++++++++++++---------
1 file changed, 41 insertions(+), 13 deletions(-)
diff --git a/subworkflows/03_filtering.nf b/subworkflows/03_filtering.nf
index dab2324..97399ef 100644
--- a/subworkflows/03_filtering.nf
+++ b/subworkflows/03_filtering.nf
@@ -46,14 +46,16 @@ workflow STEP_03_ASSEMBLY_FILTER {
with: !it[1].isEmpty()
}
- // discarded_contigs_category.view()
- samples_with_discarded_contigs = discarded_contigs_category.with
- samples_without_discarded_contigs = discarded_contigs_category.without
+
+ samples_without_discarded_contigs = discarded_contigs_category.without.map{ it -> it[0]}
+ samples_without_discarded_contigs.view{"samples WITHOUT DISCARDED CONTIGS: $it"}
+ ch_bam_of_sample_without_discarded_contigs = samples_without_discarded_contigs.join(bam)
+ ch_bam_of_sample_without_discarded_contigs.view{"samples WITHOUT DISCARDED CONTIGS AND bam: $it"}
discarded_contigs_category.without.view { "WITHOUT::::::::: > $it" }
discarded_contigs_category.with.view { "WITH::::::::: > $it" }
- ch_discarded_contigs_and_bam = samples_with_discarded_contigs.join(bam)
+ ch_discarded_contigs_and_bam = discarded_contigs_category.with.join(bam)
ch_discarded_contigs_and_bam.view()
FILTER_BAM(ch_discarded_contigs_and_bam)
@@ -79,17 +81,24 @@ workflow STEP_03_ASSEMBLY_FILTER {
}
- // ch_merged_bam = MERGE_BAM_FILES(ch_filtered_bam, ch_bam_with_discarded_reads)
- // SAMTOOLS(ch_merged_bam, '03_filtering/')
+ // remove bai from ch_bam_with_discarded_reads
+ ch_bam_to_merge = ch_bam_with_discarded_reads.map{ it -> [it[0], it[1]]}.join(ch_selected_bam)
+
+
+
+
+
+ ch_merged_bam = MERGE_BAM_FILES(ch_bam_to_merge)
+ ch_final_bam = ch_bam_of_sample_without_discarded_contigs.mix(ch_merged_bam)
- ch_merged_bam = Channel.empty()
- ch_coverage = Channel.empty()
- ch_flagstat= Channel.empty()
+ ch_final_bam.view { "FINAL BAMMMM::::::::: > $it" }
- // ch_flagstat = SAMTOOLS.out.sam_flagstat
- // ch_coverage = SAMTOOLS.out.sam_coverage
+ SAMTOOLS(ch_final_bam, '03_filtering/')
+
+ ch_flagstat = SAMTOOLS.out.sam_flagstat
+ ch_coverage = SAMTOOLS.out.sam_coverage
QUAST( ch_merged_selected, "03_filtering/quast_filtered" )
ch_quast_report = QUAST.out.report
@@ -97,7 +106,7 @@ workflow STEP_03_ASSEMBLY_FILTER {
emit:
selected_contigs = ch_merged_selected
quast_report = ch_quast_report
- bam = ch_merged_bam
+ bam = ch_final_bam
sam_coverage = ch_coverage
sam_flagstat = ch_flagstat
@@ -158,7 +167,26 @@ process EXTRACT_SINGLE_READS {
script:
"""
- samtools fastq -@ ${task.cpus} -N -1 reads.fastq.gz $bam
+ samtools fastq -@ ${task.cpus} -o reads.fastq.gz $bam
+
+ """
+}
+
+
+
+process MERGE_BAM_FILES {
+ tag "${meta.id}"
+
+ input:
+ tuple val(meta), path(bam1), path(bam2)
+
+ output:
+ tuple val(meta), path("${meta.id}.filtered.bam"), path("${meta.id}.filtered.bam.bai") , emit: bam
+
+ script:
+ """
+ samtools merge -@ ${task.cpus} -o ${meta.id}.filtered.bam $bam1 $bam2
+ samtools index -@ ${task.cpus} ${meta.id}.filtered.bam
"""
}
--
GitLab
From 0ea7819f47e0789e865304d0f9e40d967e90df81 Mon Sep 17 00:00:00 2001
From: Jean Mainguy <jean.mainguy@inra.fr>
Date: Tue, 25 Oct 2022 16:29:17 +0200
Subject: [PATCH 069/194] change name of input as it can colapse with output
name
---
modules/assembly.nf | 4 ++--
1 file changed, 2 insertions(+), 2 deletions(-)
diff --git a/modules/assembly.nf b/modules/assembly.nf
index 41db2c5..9155270 100644
--- a/modules/assembly.nf
+++ b/modules/assembly.nf
@@ -113,14 +113,14 @@ process RENAME_CONTIGS {
label 'PYTHON'
input:
- tuple val(meta), path(assembly)
+ tuple val(meta), path("${meta.id}.raw.fna")
output:
tuple val(meta), path("${meta.id}.fna"), emit: fna
script:
"""
- rename_contigs.py --sample ${meta.id} --fna_file $assembly --out_fna ${meta.id}.fna -v
+ rename_contigs.py --sample ${meta.id} --fna_file ${meta.id}.raw.fna --out_fna ${meta.id}.fna -v
"""
}
\ No newline at end of file
--
GitLab
From cc57f76ff9254f3ea5c1181a126168bfc2fe540f Mon Sep 17 00:00:00 2001
From: Jean Mainguy <jean.mainguy@inra.fr>
Date: Tue, 25 Oct 2022 16:34:26 +0200
Subject: [PATCH 070/194] add verbose parameter to filter_contig_per_cpm.py
script
---
modules/filtering_cpm.nf | 2 +-
1 file changed, 1 insertion(+), 1 deletion(-)
diff --git a/modules/filtering_cpm.nf b/modules/filtering_cpm.nf
index 0155034..cdfe8e5 100644
--- a/modules/filtering_cpm.nf
+++ b/modules/filtering_cpm.nf
@@ -12,7 +12,7 @@ process CHUNK_ASSEMBLY_FILTER {
script:
chunk_name = assembly_file.baseName
"""
- filter_contig_per_cpm.py -i ${idxstats} -f ${assembly_file} -c ${min_cpm} -s ${chunk_name}_select_cpm${min_cpm}.fasta -d ${chunk_name}_discard_cpm${min_cpm}.fasta
+ filter_contig_per_cpm.py -v -i ${idxstats} -f ${assembly_file} -c ${min_cpm} -s ${chunk_name}_select_cpm${min_cpm}.fasta -d ${chunk_name}_discard_cpm${min_cpm}.fasta
"""
}
--
GitLab
From 23f06b36a8a347e73d718d4b8a5d03169d65358d Mon Sep 17 00:00:00 2001
From: Jean Mainguy <jean.mainguy@inra.fr>
Date: Tue, 25 Oct 2022 16:35:20 +0200
Subject: [PATCH 071/194] publish bam in reads deduplication
---
modules/reads_deduplication.nf | 1 +
1 file changed, 1 insertion(+)
diff --git a/modules/reads_deduplication.nf b/modules/reads_deduplication.nf
index 4731f9b..bce077d 100644
--- a/modules/reads_deduplication.nf
+++ b/modules/reads_deduplication.nf
@@ -2,6 +2,7 @@ process READS_DEDUPLICATION {
tag "${meta.id}"
publishDir "${params.outdir}/02_assembly/deduplicated_reads/", mode: 'copy', pattern: '*.fastq.gz'
publishDir "${params.outdir}/02_assembly/${meta.id}/", mode: 'copy', pattern: '*.fastq.gz'
+ publishDir "${params.outdir}/02_assembly/${meta.id}/", mode: 'copy', pattern: '*.bam*'
input:
tuple val(meta), path(assembly), path(reads)
--
GitLab
From 289b3098bbe3f45b56e1d04dbb623ffbc073fdff Mon Sep 17 00:00:00 2001
From: Jean Mainguy <jean.mainguy@inra.fr>
Date: Tue, 25 Oct 2022 16:41:10 +0200
Subject: [PATCH 072/194] adjust output of S03_FILTERING
---
main.nf | 7 ++++---
1 file changed, 4 insertions(+), 3 deletions(-)
diff --git a/main.nf b/main.nf
index 2c53770..48338b6 100644
--- a/main.nf
+++ b/main.nf
@@ -418,7 +418,7 @@ workflow {
}
if ( !params.stop_at_clean && !params.stop_at_assembly && !params.skip_filtering ) {
- print("IN FILTERING")
+
ch_min_contigs_cpm = Channel.value(params.min_contigs_cpm)
@@ -432,9 +432,10 @@ workflow {
ch_assembly = S03_FILTERING.out.selected_contigs
ch_bam = S03_FILTERING.out.bam
- // ch_sam_coverage = S03_FILTERING.out.coverage
+
+ ch_sam_coverage = S03_FILTERING.out.sam_coverage
ch_quast_after_filter_report = S03_FILTERING.out.quast_report
- // ch_final_assembly_flagstat_report = S03_FILTERING.out.sam_flagstat
+ ch_final_assembly_flagstat_report = S03_FILTERING.out.sam_flagstat
}
--
GitLab
From b3968a1ee55a30cf4b5209eab3538fc9b6286806 Mon Sep 17 00:00:00 2001
From: Jean Mainguy <jean.mainguy@inra.fr>
Date: Tue, 25 Oct 2022 17:33:14 +0200
Subject: [PATCH 073/194] cleaning and restructuration of samtools process
---
main.nf | 11 +--
modules/read_alignment_manipulation.nf | 104 ++++++++++++++++++++++++
modules/read_alignment_metrics.nf | 24 ------
subworkflows/02_assembly.nf | 15 ++--
subworkflows/03_filtering.nf | 105 +++----------------------
subworkflows/05_alignment.nf | 27 -------
subworkflows/08_binning.nf | 8 +-
7 files changed, 125 insertions(+), 169 deletions(-)
create mode 100644 modules/read_alignment_manipulation.nf
delete mode 100644 modules/read_alignment_metrics.nf
diff --git a/main.nf b/main.nf
index 48338b6..cdd2d19 100644
--- a/main.nf
+++ b/main.nf
@@ -453,20 +453,13 @@ workflow {
ch_software_versions_S04 = S04_STRUCTURAL_ANNOT.out.software_versions
}
- // ch_bam = Channel.empty()
ch_diamond_result = Channel.empty()
- // ch_sam_coverage = Channel.empty()
if ( !params.stop_at_clean && !params.stop_at_assembly && !params.stop_at_filtering && !params.stop_at_structural_annot ) {
S05_ALIGNMENT (ch_annotation_faa, ch_diamon_db)
- // ch_bam = S05_ALIGNMENT.out.bam
+
ch_diamond_result = S05_ALIGNMENT.out.m8
- // ch_sam_coverage = S05_ALIGNMENT.out.sam_coverage
- // if (!params.skip_filtering){
- // // when filtering is skip reads vs assembly remain unchanged so no need to send it to multiqc
- // // ch_final_assembly_flagstat_report = S05_ALIGNMENT.out.sam_flagstat
- // }
ch_software_versions_S05 = S05_ALIGNMENT.out.software_versions
}
@@ -486,7 +479,7 @@ workflow {
}
if ( !params.stop_at_clean && !params.stop_at_assembly && !params.stop_at_filtering && !params.stop_at_structural_annot && !params.skip_binning ) {
- S08_BINNING( ch_reads, ch_annotation_fna, ch_bam, ch_gtbdtk_db, ch_quast )
+ S08_BINNING( ch_reads, ch_assembly, ch_bam, ch_gtbdtk_db, ch_quast )
ch_bins_abundances_report = S08_BINNING.out.bins_abundances_report
ch_bins_stats_report = S08_BINNING.out.bins_stats_report
diff --git a/modules/read_alignment_manipulation.nf b/modules/read_alignment_manipulation.nf
new file mode 100644
index 0000000..ca0fe5d
--- /dev/null
+++ b/modules/read_alignment_manipulation.nf
@@ -0,0 +1,104 @@
+process GET_ALIGNMENT_METRICS {
+ tag "${meta.id}"
+ publishDir "${params.outdir}/$publishDir_path/${meta.id}", mode: 'copy'
+
+ input:
+ tuple val(meta), path(bam), path(bai)
+ val(publishDir_path)
+
+ output:
+ tuple val(meta), path("${meta.id}.coverage.tsv"), emit: sam_coverage
+ tuple val(meta), path("${meta.id}.idxstats"), emit: sam_idxstat
+ path "${meta.id}.flagstat", emit: sam_flagstat
+ path "${meta.id}*"
+ path "v_samtools.txt", emit: v_samtools
+
+ script:
+ """
+ samtools flagstat -@ ${task.cpus} ${bam} > ${meta.id}.flagstat
+ samtools coverage ${bam} > ${meta.id}.coverage.tsv
+ samtools idxstats ${bam} > ${meta.id}.idxstats
+
+ samtools --version &> v_samtools.txt
+ """
+}
+
+
+process FILTER_BAM {
+ tag "${meta.id}"
+
+ input:
+ tuple val(meta), path(discarded_contigs), path(bam), path(bai)
+
+ output:
+ tuple val(meta), path("selected_contigs.bam"), emit: selected_contigs_bam
+ tuple val(meta), path("discarded_contigs.bam"), emit: discarded_contigs_bam
+
+
+ script:
+ """
+
+ samtools faidx $discarded_contigs
+ awk 'BEGIN {FS="\\t"}; {print \$1 FS "0" FS \$2}' ${discarded_contigs}.fai > discarded_contigs.bed
+
+
+ samtools view -@ ${task.cpus} -bL discarded_contigs.bed -U selected_contigs.bam $bam -o discarded_contigs.bam
+
+
+ """
+}
+
+process EXTRACT_PAIRED_READS {
+ tag "${meta.id}"
+
+ input:
+ tuple val(meta), path(bam)
+
+ output:
+ tuple val(meta), path("reads*.fastq.gz"), emit: reads
+
+
+ script:
+ """
+
+ samtools fastq -@ ${task.cpus} -N -1 reads_R1.fastq.gz -2 reads_R2.fastq.gz $bam
+
+ """
+}
+
+process EXTRACT_SINGLE_READS {
+ tag "${meta.id}"
+
+ input:
+ tuple val(meta), path(bam)
+
+ output:
+ tuple val(meta), path("reads.fastq.gz"), emit: reads
+
+ script:
+ """
+
+ samtools fastq -@ ${task.cpus} -o reads.fastq.gz $bam
+
+ """
+}
+
+
+
+process MERGE_BAM_FILES {
+ tag "${meta.id}"
+
+ input:
+ tuple val(meta), path(bam1), path(bam2)
+
+ output:
+ tuple val(meta), path("${meta.id}.filtered.bam"), path("${meta.id}.filtered.bam.bai") , emit: bam
+
+ script:
+ """
+ samtools merge -@ ${task.cpus} -o ${meta.id}.filtered.bam $bam1 $bam2
+ samtools index -@ ${task.cpus} ${meta.id}.filtered.bam
+
+ """
+}
+
diff --git a/modules/read_alignment_metrics.nf b/modules/read_alignment_metrics.nf
deleted file mode 100644
index ee1bd54..0000000
--- a/modules/read_alignment_metrics.nf
+++ /dev/null
@@ -1,24 +0,0 @@
-process SAMTOOLS {
- tag "${meta.id}"
- publishDir "${params.outdir}/$publishDir_path/${meta.id}", mode: 'copy'
-
- input:
- tuple val(meta), path(bam), path(bai)
- val(publishDir_path)
-
- output:
- tuple val(meta), path("${meta.id}.coverage.tsv"), emit: sam_coverage
- tuple val(meta), path("${meta.id}.idxstats"), emit: sam_idxstat
- path "${meta.id}.flagstat", emit: sam_flagstat
- path "${meta.id}*"
- path "v_samtools.txt", emit: v_samtools
-
- script:
- """
- samtools flagstat -@ ${task.cpus} ${bam} > ${meta.id}.flagstat
- samtools coverage ${bam} > ${meta.id}.coverage.tsv
- samtools idxstats ${bam} > ${meta.id}.idxstats
-
- samtools --version &> v_samtools.txt
- """
-}
\ No newline at end of file
diff --git a/subworkflows/02_assembly.nf b/subworkflows/02_assembly.nf
index ee5a207..fe90d44 100644
--- a/subworkflows/02_assembly.nf
+++ b/subworkflows/02_assembly.nf
@@ -2,7 +2,7 @@ include { METASPADES; MEGAHIT; HIFIASM_META; METAFLYE; RENAME_CONTIGS } from '..
include { QUAST as ASSEMBLY_QUAST} from '../modules/metaquast'
include { READS_DEDUPLICATION } from '../modules/reads_deduplication'
include { MINIMAP2 } from '../modules/read_alignment'
-include { SAMTOOLS } from '../modules/read_alignment_metrics'
+include { GET_ALIGNMENT_METRICS } from '../modules/read_alignment_manipulation'
workflow STEP_02_ASSEMBLY {
take:
@@ -67,22 +67,19 @@ workflow STEP_02_ASSEMBLY {
ch_bwa_mem_v = READS_DEDUPLICATION.out.v_bwa_mem2
} else {
-
- if (!params.skip_filtering) {
MINIMAP2(ch_contigs_and_reads,"02_assembly")
ch_bam = MINIMAP2.out.bam
ch_minimap2_v = MINIMAP2.out.v_minimap2
- }
}
- SAMTOOLS(ch_bam, "02_assembly")
+ GET_ALIGNMENT_METRICS(ch_bam, "02_assembly")
- ch_idxstats = SAMTOOLS.out.sam_idxstat
- ch_flagstat = SAMTOOLS.out.sam_flagstat
- ch_coverage = SAMTOOLS.out.sam_coverage
- ch_samtools_v = SAMTOOLS.out.v_samtools
+ ch_idxstats = GET_ALIGNMENT_METRICS.out.sam_idxstat
+ ch_flagstat = GET_ALIGNMENT_METRICS.out.sam_flagstat
+ ch_coverage = GET_ALIGNMENT_METRICS.out.sam_coverage
+ ch_samtools_v = GET_ALIGNMENT_METRICS.out.v_samtools
ch_software_versions = ch_assembler_v.first().mix( ch_quast_v.first(),
ch_bwa_mem_v.first(),
diff --git a/subworkflows/03_filtering.nf b/subworkflows/03_filtering.nf
index 97399ef..be38cd6 100644
--- a/subworkflows/03_filtering.nf
+++ b/subworkflows/03_filtering.nf
@@ -1,7 +1,7 @@
include { CHUNK_ASSEMBLY_FILTER; MERGE_ASSEMBLY_FILTER} from '../modules/filtering_cpm.nf'
include { QUAST } from '../modules/metaquast'
include { MINIMAP2; BWA_MEM } from '../modules/read_alignment'
-include { SAMTOOLS } from '../modules/read_alignment_metrics'
+include { GET_ALIGNMENT_METRICS; FILTER_BAM; EXTRACT_SINGLE_READS; EXTRACT_PAIRED_READS; MERGE_BAM_FILES} from '../modules/read_alignment_manipulation'
workflow STEP_03_ASSEMBLY_FILTER {
take:
@@ -41,6 +41,9 @@ workflow STEP_03_ASSEMBLY_FILTER {
ch_merged_selected = MERGE_ASSEMBLY_FILTER.out.merged_selected
discarded_contigs = MERGE_ASSEMBLY_FILTER.out.merged_discarded
+
+ // Differentiate sample with and without discarded_contigs
+ // samples with no discarded_contigs are not going to be processed to process
discarded_contigs_category = discarded_contigs.branch{
without: it[1].isEmpty()
with: !it[1].isEmpty()
@@ -48,15 +51,9 @@ workflow STEP_03_ASSEMBLY_FILTER {
samples_without_discarded_contigs = discarded_contigs_category.without.map{ it -> it[0]}
- samples_without_discarded_contigs.view{"samples WITHOUT DISCARDED CONTIGS: $it"}
ch_bam_of_sample_without_discarded_contigs = samples_without_discarded_contigs.join(bam)
- ch_bam_of_sample_without_discarded_contigs.view{"samples WITHOUT DISCARDED CONTIGS AND bam: $it"}
-
- discarded_contigs_category.without.view { "WITHOUT::::::::: > $it" }
- discarded_contigs_category.with.view { "WITH::::::::: > $it" }
ch_discarded_contigs_and_bam = discarded_contigs_category.with.join(bam)
- ch_discarded_contigs_and_bam.view()
FILTER_BAM(ch_discarded_contigs_and_bam)
@@ -75,30 +72,24 @@ workflow STEP_03_ASSEMBLY_FILTER {
} else {
ch_discarded_reads = EXTRACT_SINGLE_READS(ch_discarded_bam)
ch_contigs_and_discarded_reads = ch_merged_selected.join(ch_discarded_reads)
- MINIMAP2(ch_contigs_and_reads, "03_filtering")
+ MINIMAP2(ch_contigs_and_discarded_reads, "03_filtering")
ch_bam_with_discarded_reads = MINIMAP2.out.bam
}
- // remove bai from ch_bam_with_discarded_reads
+ // remove bai from ch_bam_with_discarded_reads and add bam of selected contigs
ch_bam_to_merge = ch_bam_with_discarded_reads.map{ it -> [it[0], it[1]]}.join(ch_selected_bam)
-
-
-
-
ch_merged_bam = MERGE_BAM_FILES(ch_bam_to_merge)
ch_final_bam = ch_bam_of_sample_without_discarded_contigs.mix(ch_merged_bam)
- ch_final_bam.view { "FINAL BAMMMM::::::::: > $it" }
-
- SAMTOOLS(ch_final_bam, '03_filtering/')
+ GET_ALIGNMENT_METRICS(ch_final_bam, '03_filtering/')
- ch_flagstat = SAMTOOLS.out.sam_flagstat
- ch_coverage = SAMTOOLS.out.sam_coverage
+ ch_flagstat = GET_ALIGNMENT_METRICS.out.sam_flagstat
+ ch_coverage = GET_ALIGNMENT_METRICS.out.sam_coverage
QUAST( ch_merged_selected, "03_filtering/quast_filtered" )
ch_quast_report = QUAST.out.report
@@ -112,81 +103,3 @@ workflow STEP_03_ASSEMBLY_FILTER {
}
-
-process FILTER_BAM {
- tag "${meta.id}"
-
- input:
- tuple val(meta), path(discarded_contigs), path(bam), path(bai)
-
- output:
- tuple val(meta), path("selected_contigs.bam"), emit: selected_contigs_bam
- tuple val(meta), path("discarded_contigs.bam"), emit: discarded_contigs_bam
-
-
- script:
- """
-
- samtools faidx $discarded_contigs
- awk 'BEGIN {FS="\\t"}; {print \$1 FS "0" FS \$2}' ${discarded_contigs}.fai > discarded_contigs.bed
-
-
- samtools view -@ ${task.cpus} -bL discarded_contigs.bed -U selected_contigs.bam $bam -o discarded_contigs.bam
-
-
- """
-}
-
-process EXTRACT_PAIRED_READS {
- tag "${meta.id}"
-
- input:
- tuple val(meta), path(bam) //, path(bai)
-
- output:
- tuple val(meta), path("reads*.fastq.gz"), emit: reads
-
-
- script:
- """
-
- samtools fastq -@ ${task.cpus} -N -1 reads_R1.fastq.gz -2 reads_R2.fastq.gz $bam
-
- """
-}
-
-process EXTRACT_SINGLE_READS {
- tag "${meta.id}"
-
- input:
- tuple val(meta), path(bam), path(bai)
-
- output:
- tuple val(meta), path("reads.fastq.gz"), emit: reads
-
- script:
- """
-
- samtools fastq -@ ${task.cpus} -o reads.fastq.gz $bam
-
- """
-}
-
-
-
-process MERGE_BAM_FILES {
- tag "${meta.id}"
-
- input:
- tuple val(meta), path(bam1), path(bam2)
-
- output:
- tuple val(meta), path("${meta.id}.filtered.bam"), path("${meta.id}.filtered.bam.bai") , emit: bam
-
- script:
- """
- samtools merge -@ ${task.cpus} -o ${meta.id}.filtered.bam $bam1 $bam2
- samtools index -@ ${task.cpus} ${meta.id}.filtered.bam
-
- """
-}
diff --git a/subworkflows/05_alignment.nf b/subworkflows/05_alignment.nf
index 191d20f..9902230 100644
--- a/subworkflows/05_alignment.nf
+++ b/subworkflows/05_alignment.nf
@@ -1,37 +1,13 @@
-include { MINIMAP2; BWA_MEM } from '../modules/read_alignment'
-include { SAMTOOLS } from '../modules/read_alignment_metrics'
include { DIAMOND } from '../modules/diamond'
workflow STEP_05_ALIGNMENT {
take:
- // contigs_and_reads
prokka_faa
diamond
main:
- // ch_bwa_mem_v = Channel.empty()
- // ch_minimap2_v = Channel.empty()
- // ch_samtools_v = Channel.empty()
ch_diamond_v = Channel.empty()
- // publishDir = "05_alignment/05_1_reads_alignment_on_contigs"
- // if (params.type == 'SR') {
- // BWA_MEM(contigs_and_reads, publishDir)
- // ch_bam = BWA_MEM.out.bam
-
- // ch_bwa_mem_v = BWA_MEM.out.v_bwa_mem2
- // ch_samtools_v = BWA_MEM.out.v_samtools
- // } else {
- // MINIMAP2(contigs_and_reads, publishDir)
- // ch_bam = MINIMAP2.out.bam
-
- // ch_minimap2_v = MINIMAP2.out.v_minimap2
- // ch_samtools_v = MINIMAP2.out.v_samtools
- // }
-
- // SAMTOOLS(ch_bam, publishDir)
- // ch_sam_coverage = SAMTOOLS.out.sam_coverage
- // ch_sam_flagstat = SAMTOOLS.out.sam_flagstat
ch_m8 =Channel.empty()
if (!params.skip_func_annot || !params.skip_taxo_affi){
@@ -43,9 +19,6 @@ workflow STEP_05_ALIGNMENT {
ch_diamond_v = DIAMOND.out.v_diamond
}
emit:
- // bam = ch_bam
m8 = ch_m8
- // sam_coverage = ch_sam_coverage
software_versions = ch_diamond_v.first()
- // sam_flagstat = ch_sam_flagstat
}
diff --git a/subworkflows/08_binning.nf b/subworkflows/08_binning.nf
index 97df1a6..59b9c7b 100644
--- a/subworkflows/08_binning.nf
+++ b/subworkflows/08_binning.nf
@@ -2,7 +2,7 @@ include { GENERATE_DEPTH_FILES; METABAT2; MAXBIN2; CONCOCT; METAWRAP_REFINMENT;
include { CHECKM2 } from '../modules/checkm2'
include { DREP } from '../modules/drep'
include { BWA_MEM;BWA_MEM as BWA_MEM_CROSS_ALIGNMENT; MINIMAP2; MINIMAP2 as MINIMAP2_CROSS_ALIGNMENT } from '../modules/read_alignment'
-include { SAMTOOLS } from '../modules/read_alignment_metrics'
+include { GET_ALIGNMENT_METRICS } from '../modules/read_alignment_manipulation'
include { GTDBTK } from '../modules/gtdbtk'
include { GENOMES_ABUNDANCES_PER_SAMPLE; ADD_QUAST_INFO_TO_BINS; BINS_STATS_TO_MUTLIQC_FORMAT} from '../modules/sum_up_bins_informations'
@@ -213,11 +213,11 @@ workflow STEP_08_BINNING {
ch_collect_coverages = Channel.empty()
ch_collect_flagstats = Channel.empty()
- SAMTOOLS(ch_bam, "08_binning/08_4_mapping_on_final_bins/stats")
+ GET_ALIGNMENT_METRICS(ch_bam, "08_binning/08_4_mapping_on_final_bins/stats")
- ch_collect_coverages = SAMTOOLS.out.sam_coverage.map {id, file -> file}
+ ch_collect_coverages = GET_ALIGNMENT_METRICS.out.sam_coverage.map {id, file -> file}
.collect()
- ch_collect_flagstats = SAMTOOLS.out.sam_flagstat.collect()
+ ch_collect_flagstats = GET_ALIGNMENT_METRICS.out.sam_flagstat.collect()
GENOMES_ABUNDANCES_PER_SAMPLE(ch_collect_coverages, ch_collect_flagstats, \
ch_bins_drep, DREP.out.output_drep_stats, GTDBTK.out.gtdbtk_affiliations_predictions,
--
GitLab
From 734ac13e66692f25d221fb22bd2204737a2875f1 Mon Sep 17 00:00:00 2001
From: Jean Mainguy <jean.mainguy@inra.fr>
Date: Tue, 25 Oct 2022 17:33:49 +0200
Subject: [PATCH 074/194] comment prokka warning
---
docs/usage.md | 4 ++--
1 file changed, 2 insertions(+), 2 deletions(-)
diff --git a/docs/usage.md b/docs/usage.md
index 672fa3a..d0b1a4c 100644
--- a/docs/usage.md
+++ b/docs/usage.md
@@ -300,7 +300,7 @@ No parameter available for this substep.
No parameters.
**WARNING 6:** `S04_STRUCTURAL_ANNOT` step depends on `S01_CLEAN_QC`, `S02_ASSEMBLY` and `S03_FILTERING` steps (if you use it). You need to use the mandatory files of these four steps to run `S04_STRUCTURAL_ANNOT`. See [II. Input files](usage.md#ii-input-files) and **WARNINGS 1 to 6**.
-
+<!--
**WARNING 7:** if you haven't previously done `S03_FILTERING`, calculation time of `S04_STRUCTURAL_ANNOT` can be important. Some cluster queues have defined calculation time, you need to adapt the queue you use to your data.
> For example, if you are on [genologin cluster](http://bioinfo.genotoul.fr/) and you haven't done the `S03_FILTERING` step, you can write a `nextflow.config` file in your working directory containing these lines:
> ```bash
@@ -308,7 +308,7 @@ No parameters.
> queue = 'unlimitq'
> }
> ```
-> This will launch the `Prokka` command line of step `04_STRUCTURAL_ANNOT` on a calculation queue (`unlimitq`) where the job can last more than 4 days (which is not the case for the usual `workq` queue).
+> This will launch the `Prokka` command line of step `04_STRUCTURAL_ANNOT` on a calculation queue (`unlimitq`) where the job can last more than 4 days (which is not the case for the usual `workq` queue). -->
#### **`S05_ALIGNMENT` step:**
--
GitLab
From 0f0894ddcef63ed233ef17e4c388f8b3076bd389 Mon Sep 17 00:00:00 2001
From: Jean Mainguy <jean.mainguy@inra.fr>
Date: Mon, 17 Oct 2022 14:57:10 +0200
Subject: [PATCH 075/194] Add prodigal module #163
---
modules/prodigal.nf | 18 ++++++++++++++++++
1 file changed, 18 insertions(+)
create mode 100644 modules/prodigal.nf
diff --git a/modules/prodigal.nf b/modules/prodigal.nf
new file mode 100644
index 0000000..1a9946c
--- /dev/null
+++ b/modules/prodigal.nf
@@ -0,0 +1,18 @@
+process PRODIGAL {
+ tag "${meta.id}"
+
+ input:
+ tuple val(meta), file(assembly_file)
+
+ output:
+ tuple val(meta), path("PROKKA_${meta.id}"), emit: prokka_results
+ path "v_prodigal.txt", emit: v_prokka
+
+ script:
+ """
+ prodigal -i ${assembly_file} -c -p meta -f gff -a prodigal.faa -o prodigal.gff
+
+ prodigal -v 2> v_prodigal.txt
+
+ """
+}
\ No newline at end of file
--
GitLab
From e9b67fa90000c14292be17b9437b4e7abefd5797 Mon Sep 17 00:00:00 2001
From: Jean Mainguy <jean.mainguy@inra.fr>
Date: Mon, 17 Oct 2022 15:51:34 +0200
Subject: [PATCH 076/194] Add barrnap module see issue #163
---
modules/barnap.nf | 18 ++++++++++++++++++
1 file changed, 18 insertions(+)
create mode 100644 modules/barnap.nf
diff --git a/modules/barnap.nf b/modules/barnap.nf
new file mode 100644
index 0000000..0b88b76
--- /dev/null
+++ b/modules/barnap.nf
@@ -0,0 +1,18 @@
+process PRODIGAL {
+ tag "${meta.id}"
+
+ input:
+ tuple val(meta), file(assembly_file)
+
+ output:
+ tuple val(meta), path("barrnap.gff"), emit: barrnap_gff
+ path "v_barrnap.txt", emit: v_barrnap
+
+ script:
+ """
+ barrnap -o barrnap.gff --threads ${task.cpus} ${assembly_file}
+
+ barrnap --version 2> v_barrnap.txt
+
+ """
+}
--
GitLab
From e6c304f263259e16c61d742afbe29b5ee44ef229 Mon Sep 17 00:00:00 2001
From: Jean Mainguy <jean.mainguy@inra.fr>
Date: Mon, 17 Oct 2022 15:52:13 +0200
Subject: [PATCH 077/194] Fix typo in prodigal module see issue #163
---
modules/prodigal.nf | 5 +++--
1 file changed, 3 insertions(+), 2 deletions(-)
diff --git a/modules/prodigal.nf b/modules/prodigal.nf
index 1a9946c..be69d07 100644
--- a/modules/prodigal.nf
+++ b/modules/prodigal.nf
@@ -5,8 +5,9 @@ process PRODIGAL {
tuple val(meta), file(assembly_file)
output:
- tuple val(meta), path("PROKKA_${meta.id}"), emit: prokka_results
- path "v_prodigal.txt", emit: v_prokka
+ tuple val(meta), path("prodigal.gff"), emit: prodigal_gff
+ tuple val(meta), path("prodigal.faa"), emit: prodigal_faa
+ path "v_prodigal.txt", emit: v_prodigal
script:
"""
--
GitLab
From a1f559dcf8023cd15652f02045037f54cfbc425d Mon Sep 17 00:00:00 2001
From: Jean Mainguy <jean.mainguy@inra.fr>
Date: Mon, 17 Oct 2022 15:55:34 +0200
Subject: [PATCH 078/194] fix module name
---
modules/barnap.nf | 2 +-
1 file changed, 1 insertion(+), 1 deletion(-)
diff --git a/modules/barnap.nf b/modules/barnap.nf
index 0b88b76..302ab6d 100644
--- a/modules/barnap.nf
+++ b/modules/barnap.nf
@@ -1,4 +1,4 @@
-process PRODIGAL {
+process BARRNAP {
tag "${meta.id}"
input:
--
GitLab
From bc84d7ad3886d50ebeddb10f215399832c7efc6d Mon Sep 17 00:00:00 2001
From: Jean Mainguy <jean.mainguy@inra.fr>
Date: Mon, 17 Oct 2022 16:18:15 +0200
Subject: [PATCH 079/194] fix typo in barrnap name module
---
modules/{barnap.nf => barrnap.nf} | 0
1 file changed, 0 insertions(+), 0 deletions(-)
rename modules/{barnap.nf => barrnap.nf} (100%)
diff --git a/modules/barnap.nf b/modules/barrnap.nf
similarity index 100%
rename from modules/barnap.nf
rename to modules/barrnap.nf
--
GitLab
From c3e4e1e3c4ffa69484d74f4f6f4f495795012417 Mon Sep 17 00:00:00 2001
From: Jean Mainguy <jean.mainguy@inra.fr>
Date: Mon, 17 Oct 2022 17:10:09 +0200
Subject: [PATCH 080/194] structuring step 04 with prodigal + barrnap approach
---
main.nf | 17 +++++++++--------
subworkflows/04_structural_annot.nf | 26 ++++++++++++++++----------
2 files changed, 25 insertions(+), 18 deletions(-)
diff --git a/main.nf b/main.nf
index 229345d..cde0ad1 100644
--- a/main.nf
+++ b/main.nf
@@ -443,17 +443,18 @@ workflow {
if ( !params.stop_at_clean && !params.stop_at_assembly && !params.stop_at_filtering ) {
S04_STRUCTURAL_ANNOT ( ch_assembly )
- ch_prokka_ffn = S04_STRUCTURAL_ANNOT.out.ffn
- ch_prokka_faa = S04_STRUCTURAL_ANNOT.out.faa
- ch_prokka_gff = S04_STRUCTURAL_ANNOT.out.gff
- ch_prokka_fna = S04_STRUCTURAL_ANNOT.out.fna
- ch_prokka_report = S04_STRUCTURAL_ANNOT.out.report
+ // ch_prokka_ffn = S04_STRUCTURAL_ANNOT.out.ffn
+ // ch_prokka_faa = S04_STRUCTURAL_ANNOT.out.faa
+ // ch_prokka_gff = S04_STRUCTURAL_ANNOT.out.gff
+ // ch_prokka_fna = S04_STRUCTURAL_ANNOT.out.fna
+ // ch_prokka_report = S04_STRUCTURAL_ANNOT.out.report
- ch_contigs_and_reads = ch_prokka_fna
- .join(ch_reads, remainder: true)
- ch_prot_length = S04_STRUCTURAL_ANNOT.out.prot_length
+ // ch_contigs_and_reads = ch_prokka_fna
+ // .join(ch_reads, remainder: true)
+ // ch_prot_length = S04_STRUCTURAL_ANNOT.out.prot_length
ch_software_versions_S04 = S04_STRUCTURAL_ANNOT.out.software_versions
+ // exit 0, 'Exit now... S04_STRUCTURAL_ANNOT being constructed '
}
ch_bam = Channel.empty()
diff --git a/subworkflows/04_structural_annot.nf b/subworkflows/04_structural_annot.nf
index 61bff36..ba3d4fe 100644
--- a/subworkflows/04_structural_annot.nf
+++ b/subworkflows/04_structural_annot.nf
@@ -1,19 +1,25 @@
-include { PROKKA; RENAME_CONTIGS_AND_GENES } from '../modules/prokka'
+include { PRODIGAL } from '../modules/prodigal'
+include { BARRNAP } from '../modules/barrnap'
workflow STEP_04_STRUCTURAL_ANNOT {
take: assembly
main:
- PROKKA( assembly )
- ch_software_versions = PROKKA.out.v_prokka.first()
- RENAME_CONTIGS_AND_GENES(PROKKA.out.prokka_results)
+ PRODIGAL( assembly )
+ BARRNAP( assembly )
+ ch_software_versions = PRODIGAL.out.v_prodigal.first()
+
+
+ ch_software_versions = PRODIGAL.out.v_prodigal.first().mix( BARRNAP.out.v_barrnap.first())
+
+ // RENAME_CONTIGS_AND_GENES(PROKKA.out.prokka_results)
emit:
- report = PROKKA.out.report
- fna = RENAME_CONTIGS_AND_GENES.out.fna
- ffn = RENAME_CONTIGS_AND_GENES.out.ffn
- gff = RENAME_CONTIGS_AND_GENES.out.gff
- faa = RENAME_CONTIGS_AND_GENES.out.faa
- prot_length = RENAME_CONTIGS_AND_GENES.out.prot_length
+ // report = PROKKA.out.report
+ // fna = RENAME_CONTIGS_AND_GENES.out.fna
+ // ffn = RENAME_CONTIGS_AND_GENES.out.ffn
+ // gff = RENAME_CONTIGS_AND_GENES.out.gff
+ // faa = RENAME_CONTIGS_AND_GENES.out.faa
+ // prot_length = RENAME_CONTIGS_AND_GENES.out.prot_length
software_versions = ch_software_versions
}
\ No newline at end of file
--
GitLab
From 6bd03a28668b74fe8ba946c9dc577966efb273cc Mon Sep 17 00:00:00 2001
From: Jean Mainguy <jean.mainguy@inra.fr>
Date: Mon, 17 Oct 2022 17:10:43 +0200
Subject: [PATCH 081/194] fix barnnap output
---
modules/barrnap.nf | 2 +-
1 file changed, 1 insertion(+), 1 deletion(-)
diff --git a/modules/barrnap.nf b/modules/barrnap.nf
index 302ab6d..44cbd5e 100644
--- a/modules/barrnap.nf
+++ b/modules/barrnap.nf
@@ -10,7 +10,7 @@ process BARRNAP {
script:
"""
- barrnap -o barrnap.gff --threads ${task.cpus} ${assembly_file}
+ barrnap --threads ${task.cpus} ${assembly_file} > barrnap.gff
barrnap --version 2> v_barrnap.txt
--
GitLab
From bcc03469e7b55eb316b78af8e40ba591361a42ab Mon Sep 17 00:00:00 2001
From: Jean Mainguy <jean.mainguy@inra.fr>
Date: Wed, 19 Oct 2022 10:35:40 +0200
Subject: [PATCH 082/194] Add trnascan and pyfastx in conda recipe
issue #163
---
env/metagWGS.yml | 2 +-
1 file changed, 1 insertion(+), 1 deletion(-)
diff --git a/env/metagWGS.yml b/env/metagWGS.yml
index 8a0cc9e..6a0b710 100644
--- a/env/metagWGS.yml
+++ b/env/metagWGS.yml
@@ -28,5 +28,5 @@ dependencies:
- flye=2.9.1
- hifiasm_meta=hamtv0.3
- plotly
+ - trnascan-se=2.0.11
- pyfastx
-
--
GitLab
From 72e5196a0616b0f6373941b2e4a81cfe4ddcff25 Mon Sep 17 00:00:00 2001
From: Jean Mainguy <jean.mainguy@inra.fr>
Date: Wed, 19 Oct 2022 11:45:15 +0200
Subject: [PATCH 083/194] Add trnascan_se module issue #163
---
modules/trnascan_se.nf | 19 +++++++++++++++++++
subworkflows/04_structural_annot.nf | 6 +++++-
2 files changed, 24 insertions(+), 1 deletion(-)
create mode 100644 modules/trnascan_se.nf
diff --git a/modules/trnascan_se.nf b/modules/trnascan_se.nf
new file mode 100644
index 0000000..e292e3e
--- /dev/null
+++ b/modules/trnascan_se.nf
@@ -0,0 +1,19 @@
+process TRNASCAN_SE {
+ tag "${meta.id}"
+
+ input:
+ tuple val(meta), file(assembly_file)
+
+ output:
+ tuple val(meta), path("trnascan_se.gff"), emit: trnascan_gff
+ path "v_tRNAscan.txt", emit: v_tRNAscan
+
+ script:
+ """
+ tRNAscan-SE -B --gff trnascan_se.gff --thread ${task.cpus} --stats trnascan_se.log ${assembly_file}
+
+ tRNAscan-SE -h 2> v_tRNAscan.txt
+
+
+ """
+}
diff --git a/subworkflows/04_structural_annot.nf b/subworkflows/04_structural_annot.nf
index ba3d4fe..4e6687b 100644
--- a/subworkflows/04_structural_annot.nf
+++ b/subworkflows/04_structural_annot.nf
@@ -1,5 +1,6 @@
include { PRODIGAL } from '../modules/prodigal'
include { BARRNAP } from '../modules/barrnap'
+include { TRNASCAN_SE } from '../modules/trnascan_se'
workflow STEP_04_STRUCTURAL_ANNOT {
take: assembly
@@ -7,10 +8,13 @@ workflow STEP_04_STRUCTURAL_ANNOT {
main:
PRODIGAL( assembly )
BARRNAP( assembly )
+ TRNASCAN_SE( assembly )
+
ch_software_versions = PRODIGAL.out.v_prodigal.first()
- ch_software_versions = PRODIGAL.out.v_prodigal.first().mix( BARRNAP.out.v_barrnap.first())
+ ch_software_versions = PRODIGAL.out.v_prodigal.first().mix( BARRNAP.out.v_barrnap.first(),
+ TRNASCAN_SE.out.v_tRNAscan.first())
// RENAME_CONTIGS_AND_GENES(PROKKA.out.prokka_results)
--
GitLab
From 1c41a2fb681c8fe4f875b98c748d2fa182bc2a26 Mon Sep 17 00:00:00 2001
From: Jean Mainguy <jean.mainguy@inra.fr>
Date: Wed, 19 Oct 2022 17:48:21 +0200
Subject: [PATCH 084/194] simplify channel name
---
modules/barrnap.nf | 2 +-
modules/prodigal.nf | 4 ++--
modules/trnascan_se.nf | 2 +-
3 files changed, 4 insertions(+), 4 deletions(-)
diff --git a/modules/barrnap.nf b/modules/barrnap.nf
index 44cbd5e..16013a0 100644
--- a/modules/barrnap.nf
+++ b/modules/barrnap.nf
@@ -5,7 +5,7 @@ process BARRNAP {
tuple val(meta), file(assembly_file)
output:
- tuple val(meta), path("barrnap.gff"), emit: barrnap_gff
+ tuple val(meta), path("barrnap.gff"), emit: gff
path "v_barrnap.txt", emit: v_barrnap
script:
diff --git a/modules/prodigal.nf b/modules/prodigal.nf
index be69d07..63bde69 100644
--- a/modules/prodigal.nf
+++ b/modules/prodigal.nf
@@ -5,8 +5,8 @@ process PRODIGAL {
tuple val(meta), file(assembly_file)
output:
- tuple val(meta), path("prodigal.gff"), emit: prodigal_gff
- tuple val(meta), path("prodigal.faa"), emit: prodigal_faa
+ tuple val(meta), path("prodigal.gff"), emit: gff
+ tuple val(meta), path("prodigal.faa"), emit: faa
path "v_prodigal.txt", emit: v_prodigal
script:
diff --git a/modules/trnascan_se.nf b/modules/trnascan_se.nf
index e292e3e..b690c7c 100644
--- a/modules/trnascan_se.nf
+++ b/modules/trnascan_se.nf
@@ -5,7 +5,7 @@ process TRNASCAN_SE {
tuple val(meta), file(assembly_file)
output:
- tuple val(meta), path("trnascan_se.gff"), emit: trnascan_gff
+ tuple val(meta), path("trnascan_se.gff"), emit: gff
path "v_tRNAscan.txt", emit: v_tRNAscan
script:
--
GitLab
From 12c13d247568517f985d346ce0ba8a4a0d705b71 Mon Sep 17 00:00:00 2001
From: Jean Mainguy <jean.mainguy@inra.fr>
Date: Wed, 19 Oct 2022 18:03:14 +0200
Subject: [PATCH 085/194] Add process and script to merge gff annotations issue
#163
---
bin/merge_annotations.py | 178 ++++++++++++++++++++++++++++
subworkflows/04_structural_annot.nf | 12 ++
2 files changed, 190 insertions(+)
create mode 100755 bin/merge_annotations.py
diff --git a/bin/merge_annotations.py b/bin/merge_annotations.py
new file mode 100755
index 0000000..8d3b4fa
--- /dev/null
+++ b/bin/merge_annotations.py
@@ -0,0 +1,178 @@
+#!/usr/bin/env python3
+
+"""
+Combine structural annotations made from different gff.
+
+In case of collapsing annotations RNA annotation are prefered to CDS annotations.
+
+:Example:
+python template.py -v
+"""
+
+# Metadata
+__author__ = 'Mainguy Jean - Plateforme bioinformatique Toulouse'
+__copyright__ = 'Copyright (C) 2020 INRAE'
+__license__ = 'GNU General Public License'
+__version__ = '0.1'
+__email__ = 'support.bioinfo.genotoul@inra.fr'
+__status__ = 'dev'
+
+
+from argparse import ArgumentParser, ArgumentDefaultsHelpFormatter, FileType
+import logging
+import gzip
+import csv
+from collections import defaultdict
+
+def read_file_and_ignore_hashtag(file_path):
+ proper_open = gzip.open if file_path.endswith('.gz') else open
+ with proper_open(file_path, 'rt') as fl:
+ for line in fl:
+ if line.startswith('#'):
+ continue
+ yield line
+
+def group_annotations_by_contigs(*gff_annotations):
+
+ contig2annotations = defaultdict(list)
+ for annotations in gff_annotations:
+ for annotation in annotations:
+ contig2annotations[annotation['seqname']].append(annotation)
+
+ return contig2annotations
+
+
+def parse_gff_file(gff_file, feature_to_keep=False):
+
+ gff_headers = ("seqname", "_3", "feature", "start", "end", "_2", "strand", "_1", "attribute")
+
+ gff_annotations = csv.DictReader(read_file_and_ignore_hashtag(gff_file),
+ delimiter='\t',
+ fieldnames=gff_headers)
+
+ if feature_to_keep:
+ logging.info(f'Keeping only {feature_to_keep} feature from {gff_file}')
+ gff_annotations = (annotation for annotation in gff_annotations if annotation['feature'] == feature_to_keep)
+
+ return gff_annotations
+
+def parse_arguments():
+ """Parse script arguments."""
+ parser = ArgumentParser(description="...",
+ formatter_class=ArgumentDefaultsHelpFormatter)
+
+ parser.add_argument('-c', '--cds', help='gff file with CDS annotations.', required=True)
+
+ parser.add_argument('-t', '--trna', help='gff file with tRNA annotations.', required=True)
+
+ parser.add_argument('-r', '--rrna', help='gff file with rRNA annotations.', required=True)
+
+ parser.add_argument('-o', '--gff_output', help='final gff file with merged annotations.', required=True)
+
+
+ parser.add_argument("-v", "--verbose", help="increase output verbosity",
+ action="store_true")
+
+ args = parser.parse_args()
+ return args
+
+def remove_overlapping_cds(cds_file, contig2rnas):
+
+ contig_annotations = []
+ current_contig = None
+
+ for cds in parse_gff_file(cds_file, 'CDS'):
+
+ reading_next_contig = cds['seqname'] != current_contig
+
+ if contig_annotations and reading_next_contig:
+
+ yield current_contig, contig_annotations
+ contig_annotations = []
+
+ current_contig = cds['seqname']
+
+ is_overlapping = False
+ for rna in contig2rnas[current_contig]:
+ if (int(rna['end']) < int(cds['start']) or int(rna['start']) > int(cds['end'])):
+ continue
+
+ else: # overlap -> remove cds
+ is_overlapping = True
+ overlap = f"[{max(rna['start'], cds['start'])},{min(rna['end'], cds['end'])}]"
+ rna_info = f"{rna['feature']} [{rna['start']}, {rna['end']}]"
+ cds_info = f"CDS [{cds['start']}, {cds['end']}]"
+ logging.info(f"overlap: {cds_info} overlapping with {rna_info} at {overlap} on contig={current_contig}")
+
+ if not is_overlapping:
+ contig_annotations.append(cds)
+
+ yield current_contig, contig_annotations
+
+def merging_cds_and_rna(cds_per_contig, contig2rnas):
+ contig_processed = []
+ for contig, cds_features in cds_per_contig:
+ contig_processed.append(contig)
+ rna_features = contig2rnas[contig]
+ yield contig, rna_features + cds_features
+
+ # check that all rrna contigs have been processed if not processe them
+ for contig, rnas in contig2rnas.items():
+ if contig not in contig_processed:
+ logging.info(f'{contig} has only rna annotation')
+ yield contig, rnas
+
+def writing_features_to_gff(annotations_per_contig, out_gff):
+
+ with open(out_gff, 'w') as fl:
+
+ fl.write("##gff-version 3\n")
+ for contig, features in annotations_per_contig:
+ logging.info(f"writing {contig} annotations to gff file")
+ fl.write(f"##sequence-region {contig}\n")
+
+ for feature in sorted(features, key=lambda x: int(x['start'])):
+ fl.write('\t'.join(feature.values())+"\n")
+
+def main():
+
+ args = parse_arguments()
+
+ if args.verbose:
+ logging.basicConfig(format="%(levelname)s: %(message)s", level=logging.DEBUG)
+ logging.info('Mode verbose ON')
+
+ else:
+ logging.basicConfig(format="%(levelname)s: %(message)s")
+
+
+ trna_file = args.trna
+ rrna_file = args.rrna
+ cds_file = args.cds
+
+ output_gff = args.gff_output
+
+ logging.info('Parsing rRNA annoations.')
+ rrna_annotations = parse_gff_file(rrna_file, 'rRNA')
+
+ logging.info('Parsing tRNA annoations.')
+ trna_annotations = parse_gff_file(trna_file, 'tRNA')
+
+ logging.info('Grouping RNA annoations by contig.')
+ contig2rnas = group_annotations_by_contigs(trna_annotations, rrna_annotations)
+
+ logging.info('Removing CDS annoations overlapping a RNA annotations.')
+ unoverlapping_cds_per_contig = remove_overlapping_cds(cds_file, contig2rnas)
+
+ logging.info('Merge CDS and RNA annotations by contig.')
+ annotation_per_contigs = merging_cds_and_rna(unoverlapping_cds_per_contig, contig2rnas)
+
+ logging.info(f'Writting CDS and RNA annotations to gff file {output_gff}.')
+ writing_features_to_gff(annotation_per_contigs, output_gff)
+
+
+
+
+
+if __name__ == '__main__':
+ main()
\ No newline at end of file
diff --git a/subworkflows/04_structural_annot.nf b/subworkflows/04_structural_annot.nf
index 4e6687b..a70b215 100644
--- a/subworkflows/04_structural_annot.nf
+++ b/subworkflows/04_structural_annot.nf
@@ -1,6 +1,8 @@
include { PRODIGAL } from '../modules/prodigal'
include { BARRNAP } from '../modules/barrnap'
include { TRNASCAN_SE } from '../modules/trnascan_se'
+include { MERGE_ANNOTATIONS } from '../modules/merge_annotations'
+
workflow STEP_04_STRUCTURAL_ANNOT {
take: assembly
@@ -10,6 +12,16 @@ workflow STEP_04_STRUCTURAL_ANNOT {
BARRNAP( assembly )
TRNASCAN_SE( assembly )
+
+ annotations_ch = PRODIGAL.out.gff.join(BARRNAP.out.gff)
+ .join(TRNASCAN_SE.out.gff)
+ annotations_ch.view()
+
+
+
+ MERGE_ANNOTATIONS(annotations_ch)
+
+
ch_software_versions = PRODIGAL.out.v_prodigal.first()
--
GitLab
From 382a5e373a8cd4b8c0b2776596b8b58a6e755e90 Mon Sep 17 00:00:00 2001
From: Jean Mainguy <jean.mainguy@inra.fr>
Date: Wed, 19 Oct 2022 19:01:45 +0200
Subject: [PATCH 086/194] add ffn output on top of gff output
---
bin/merge_annotations.py | 51 +++++++++++++++++++++++++----
subworkflows/04_structural_annot.nf | 2 +-
2 files changed, 45 insertions(+), 8 deletions(-)
diff --git a/bin/merge_annotations.py b/bin/merge_annotations.py
index 8d3b4fa..91ebdb0 100755
--- a/bin/merge_annotations.py
+++ b/bin/merge_annotations.py
@@ -23,6 +23,8 @@ import logging
import gzip
import csv
from collections import defaultdict
+import pyfastx
+
def read_file_and_ignore_hashtag(file_path):
proper_open = gzip.open if file_path.endswith('.gz') else open
@@ -67,7 +69,11 @@ def parse_arguments():
parser.add_argument('-r', '--rrna', help='gff file with rRNA annotations.', required=True)
- parser.add_argument('-o', '--gff_output', help='final gff file with merged annotations.', required=True)
+ parser.add_argument('--gff_output', help='final gff file with all annotations.', default="all_annotation.gff")
+ parser.add_argument('--ffn_output', help='final ffn file with all annotations.', default="all_annotation.ffn")
+
+ parser.add_argument('--contig_seq', help='fasta file of contigs. Needed to extract gene annotations', required=True)
+
parser.add_argument("-v", "--verbose", help="increase output verbosity",
@@ -122,17 +128,44 @@ def merging_cds_and_rna(cds_per_contig, contig2rnas):
logging.info(f'{contig} has only rna annotation')
yield contig, rnas
-def writing_features_to_gff(annotations_per_contig, out_gff):
+def add_new_ID_tag(gff_attributes, new_id):
+ gff_attributes_no_id = ';'.join([attr for attr in gff_attributes.split(';') if attr.split('=')[0] != 'ID'])
+ return f"ID={new_id};{gff_attributes_no_id}"
+
+def get_Name_tag(gff_attributes):
+ for attr in gff_attributes.split(';'):
+ if attr.split('=')[0] == "Name":
+ return attr.split('=')[1]
+ return ''
+
+
+def writing_features_to_gff_and_ffn(annotations_per_contig, out_gff, fna_file, out_ffn):
- with open(out_gff, 'w') as fl:
+ contig_fa = pyfastx.Fasta(fna_file)
+
+
+ with open(out_gff, 'w') as fh_gff, open(out_ffn, 'w') as fh_ffn:
- fl.write("##gff-version 3\n")
+ fh_gff.write("##gff-version 3\n")
for contig, features in annotations_per_contig:
+ gene_count = defaultdict(int)
logging.info(f"writing {contig} annotations to gff file")
- fl.write(f"##sequence-region {contig}\n")
+ fh_gff.write(f"##sequence-region {contig}\n")
for feature in sorted(features, key=lambda x: int(x['start'])):
- fl.write('\t'.join(feature.values())+"\n")
+ gene_count[feature['feature']] += 1
+ new_id = f"{feature['seqname']}.{feature['feature']}_{gene_count[feature['feature']]}"
+
+ feature['attribute'] = add_new_ID_tag(feature['attribute'], new_id)
+
+ tag_name = get_Name_tag(feature['attribute'])
+ fh_gff.write('\t'.join(feature.values())+"\n")
+
+ feature_seq = contig_fa.fetch(contig, [int(feature['start']), int(feature['end'])])
+ fh_ffn.write(f">{new_id} {tag_name}\n")
+ fh_ffn.write(f"{feature_seq}\n")
+
+
def main():
@@ -152,6 +185,9 @@ def main():
output_gff = args.gff_output
+ fna_file = args.contig_seq
+ out_ffn = args.ffn_output
+
logging.info('Parsing rRNA annoations.')
rrna_annotations = parse_gff_file(rrna_file, 'rRNA')
@@ -168,11 +204,12 @@ def main():
annotation_per_contigs = merging_cds_and_rna(unoverlapping_cds_per_contig, contig2rnas)
logging.info(f'Writting CDS and RNA annotations to gff file {output_gff}.')
- writing_features_to_gff(annotation_per_contigs, output_gff)
+ writing_features_to_gff_and_ffn(annotation_per_contigs, output_gff, fna_file, out_ffn)
+
if __name__ == '__main__':
main()
\ No newline at end of file
diff --git a/subworkflows/04_structural_annot.nf b/subworkflows/04_structural_annot.nf
index a70b215..e420d55 100644
--- a/subworkflows/04_structural_annot.nf
+++ b/subworkflows/04_structural_annot.nf
@@ -13,7 +13,7 @@ workflow STEP_04_STRUCTURAL_ANNOT {
TRNASCAN_SE( assembly )
- annotations_ch = PRODIGAL.out.gff.join(BARRNAP.out.gff)
+ annotations_ch = assembly.join(PRODIGAL.out.gff).join(BARRNAP.out.gff)
.join(TRNASCAN_SE.out.gff)
annotations_ch.view()
--
GitLab
From 4cfbd05d10dda1be0fe1d2c3114d87e37e494782 Mon Sep 17 00:00:00 2001
From: Jean Mainguy <jean.mainguy@inra.fr>
Date: Thu, 20 Oct 2022 09:31:10 +0200
Subject: [PATCH 087/194] process to launch python script to merge gff files
---
modules/merge_annotations.nf | 15 +++++++++++++++
1 file changed, 15 insertions(+)
create mode 100644 modules/merge_annotations.nf
diff --git a/modules/merge_annotations.nf b/modules/merge_annotations.nf
new file mode 100644
index 0000000..4cbbd82
--- /dev/null
+++ b/modules/merge_annotations.nf
@@ -0,0 +1,15 @@
+process MERGE_ANNOTATIONS {
+ tag "${meta.id}"
+
+ input:
+ tuple val(meta), file(assembly_file), file(cds_gff), file(rrna_gff), file(trna_gff)
+
+ output:
+
+
+ script:
+ """
+ merge_annotations.py -c $cds_gff -r $rrna_gff -t $trna_gff -v --contig_seq $assembly_file \
+ --ffn_output ${meta.sample}.ffn --gff_output ${meta.sample}.gff
+ """
+}
\ No newline at end of file
--
GitLab
From ed9bd2fe5525554124aada4033dbd2b187655976 Mon Sep 17 00:00:00 2001
From: Jean Mainguy <jean.mainguy@inra.fr>
Date: Thu, 20 Oct 2022 13:37:32 +0200
Subject: [PATCH 088/194] fix strand and zero base position when extracting seq
---
bin/merge_annotations.py | 112 +++++++++++++++++++++++++--------------
1 file changed, 73 insertions(+), 39 deletions(-)
diff --git a/bin/merge_annotations.py b/bin/merge_annotations.py
index 91ebdb0..5434cdb 100755
--- a/bin/merge_annotations.py
+++ b/bin/merge_annotations.py
@@ -24,9 +24,36 @@ import gzip
import csv
from collections import defaultdict
import pyfastx
+from Bio.Seq import Seq
+def parse_arguments():
+ """Parse script arguments."""
+ parser = ArgumentParser(description="...",
+ formatter_class=ArgumentDefaultsHelpFormatter)
+
+ parser.add_argument('-c', '--cds', help='gff file with CDS annotations.', required=True)
+
+ parser.add_argument('-t', '--trna', help='gff file with tRNA annotations.', required=True)
+
+ parser.add_argument('-r', '--rrna', help='gff file with rRNA annotations.', required=True)
+
+ parser.add_argument('--gff_output', help='final gff file with all annotations.', default="all_annotation.gff")
+
+ parser.add_argument('--ffn_output', help='final ffn file with all annotations.', default="all_annotation.ffn")
+
+ parser.add_argument('--faa_output', help='final faa file with all CDS annotations in amino acid.', default="all_annotation.faa")
+
+ parser.add_argument('--contig_seq', help='fasta file of contigs. Needed to extract gene annotations', required=True)
+
+ parser.add_argument("-v", "--verbose", help="increase output verbosity",
+ action="store_true")
+
+ args = parser.parse_args()
+ return args
+
def read_file_and_ignore_hashtag(file_path):
+
proper_open = gzip.open if file_path.endswith('.gz') else open
with proper_open(file_path, 'rt') as fl:
for line in fl:
@@ -58,30 +85,6 @@ def parse_gff_file(gff_file, feature_to_keep=False):
return gff_annotations
-def parse_arguments():
- """Parse script arguments."""
- parser = ArgumentParser(description="...",
- formatter_class=ArgumentDefaultsHelpFormatter)
-
- parser.add_argument('-c', '--cds', help='gff file with CDS annotations.', required=True)
-
- parser.add_argument('-t', '--trna', help='gff file with tRNA annotations.', required=True)
-
- parser.add_argument('-r', '--rrna', help='gff file with rRNA annotations.', required=True)
-
- parser.add_argument('--gff_output', help='final gff file with all annotations.', default="all_annotation.gff")
- parser.add_argument('--ffn_output', help='final ffn file with all annotations.', default="all_annotation.ffn")
-
- parser.add_argument('--contig_seq', help='fasta file of contigs. Needed to extract gene annotations', required=True)
-
-
-
- parser.add_argument("-v", "--verbose", help="increase output verbosity",
- action="store_true")
-
- args = parser.parse_args()
- return args
-
def remove_overlapping_cds(cds_file, contig2rnas):
contig_annotations = []
@@ -116,6 +119,7 @@ def remove_overlapping_cds(cds_file, contig2rnas):
yield current_contig, contig_annotations
def merging_cds_and_rna(cds_per_contig, contig2rnas):
+
contig_processed = []
for contig, cds_features in cds_per_contig:
contig_processed.append(contig)
@@ -129,21 +133,22 @@ def merging_cds_and_rna(cds_per_contig, contig2rnas):
yield contig, rnas
def add_new_ID_tag(gff_attributes, new_id):
+
gff_attributes_no_id = ';'.join([attr for attr in gff_attributes.split(';') if attr.split('=')[0] != 'ID'])
return f"ID={new_id};{gff_attributes_no_id}"
-def get_Name_tag(gff_attributes):
- for attr in gff_attributes.split(';'):
- if attr.split('=')[0] == "Name":
+
+def get_tag_value(gff_attribute, tag):
+
+ for attr in gff_attribute.split(';'):
+ if attr.split('=')[0] == tag:
return attr.split('=')[1]
return ''
-
def writing_features_to_gff_and_ffn(annotations_per_contig, out_gff, fna_file, out_ffn):
contig_fa = pyfastx.Fasta(fna_file)
-
with open(out_gff, 'w') as fh_gff, open(out_ffn, 'w') as fh_ffn:
fh_gff.write("##gff-version 3\n")
@@ -158,13 +163,41 @@ def writing_features_to_gff_and_ffn(annotations_per_contig, out_gff, fna_file, o
feature['attribute'] = add_new_ID_tag(feature['attribute'], new_id)
- tag_name = get_Name_tag(feature['attribute'])
+ tag_name = get_tag_value(feature['attribute'], 'Name')
fh_gff.write('\t'.join(feature.values())+"\n")
- feature_seq = contig_fa.fetch(contig, [int(feature['start']), int(feature['end'])])
- fh_ffn.write(f">{new_id} {tag_name}\n")
- fh_ffn.write(f"{feature_seq}\n")
+ # feature_seq = contig_fa.fetch(contig, [int(feature['start']), int(feature['end'])])
+ #
+
+ start_zero, end_zero = int(feature['start'])-1, int(feature['end'])
+
+ if feature['strand'] == "+":
+ feature_seq = contig_fa[contig][start_zero:end_zero].seq
+ else:
+ # reverse complement the sequence
+ feature_seq = contig_fa[contig][start_zero:end_zero].antisense
+
+ fh_ffn.write(f">{new_id} {tag_name}\n{feature_seq}\n")
+
+
+ # if feature['feature'] == "CDS":
+
+ # aa_seq = Seq(feature_seq).translate(table=translation_table, cds=True)
+
+ # fh_faa.write(f'>{new_id}\n{aa_seq}\n')
+
+
+# def filter_faa_file(annotation_per_contigs, faa_file, out_faa):
+
+# protein_fa = pyfastx.Fasta(faa_file)
+# all_names = [set(protein_fa.keys().filter())]
+
+
+# for contig, features in annotation_per_contigs:
+
+
+# yield contig, features
def main():
@@ -187,6 +220,9 @@ def main():
fna_file = args.contig_seq
out_ffn = args.ffn_output
+ out_faa = args.faa_output
+
+ faa_file = "prodigal.faa"
logging.info('Parsing rRNA annoations.')
rrna_annotations = parse_gff_file(rrna_file, 'rRNA')
@@ -203,13 +239,11 @@ def main():
logging.info('Merge CDS and RNA annotations by contig.')
annotation_per_contigs = merging_cds_and_rna(unoverlapping_cds_per_contig, contig2rnas)
- logging.info(f'Writting CDS and RNA annotations to gff file {output_gff}.')
- writing_features_to_gff_and_ffn(annotation_per_contigs, output_gff, fna_file, out_ffn)
-
-
-
-
+ # annotation_per_contigs = filter_faa_file(annotation_per_contigs, faa_file, out_faa)
+ logging.info(f'Writting CDS and RNA annotations to gff file: {output_gff}.')
+ writing_features_to_gff_and_ffn(annotation_per_contigs, output_gff, fna_file, out_ffn) #, out_faa)
+
if __name__ == '__main__':
main()
\ No newline at end of file
--
GitLab
From b916348db7b64d2359353752bf2b5046608e4354 Mon Sep 17 00:00:00 2001
From: Jean Mainguy <jean.mainguy@inra.fr>
Date: Thu, 20 Oct 2022 14:35:49 +0200
Subject: [PATCH 089/194] add faa file filtering from prodigal
---
bin/merge_annotations.py | 69 +++++++++++++++++++++++++---------------
1 file changed, 44 insertions(+), 25 deletions(-)
diff --git a/bin/merge_annotations.py b/bin/merge_annotations.py
index 5434cdb..e6a5c59 100755
--- a/bin/merge_annotations.py
+++ b/bin/merge_annotations.py
@@ -145,11 +145,13 @@ def get_tag_value(gff_attribute, tag):
return attr.split('=')[1]
return ''
-def writing_features_to_gff_and_ffn(annotations_per_contig, out_gff, fna_file, out_ffn):
+def writing_features_to_gff_ffn_faa(annotations_per_contig, out_gff, fna_file, out_ffn, faa_file, out_faa):
contig_fa = pyfastx.Fasta(fna_file)
+ protein_fa = pyfastx.Fasta(faa_file)
- with open(out_gff, 'w') as fh_gff, open(out_ffn, 'w') as fh_ffn:
+
+ with open(out_gff, 'w') as fh_gff, open(out_ffn, 'w') as fh_ffn, open(out_faa, 'w') as fh_faa:
fh_gff.write("##gff-version 3\n")
for contig, features in annotations_per_contig:
@@ -161,43 +163,60 @@ def writing_features_to_gff_and_ffn(annotations_per_contig, out_gff, fna_file, o
gene_count[feature['feature']] += 1
new_id = f"{feature['seqname']}.{feature['feature']}_{gene_count[feature['feature']]}"
+ start, end = int(feature['start']), int(feature['end'])
+
+ # write faa
+ if feature['feature'] == "CDS":
+ gff_id = get_tag_value(feature['attribute'], 'ID')
+
+ # ID in gff from prodigal is <contig_number>_<cds_number>
+ # seq name in faa from prodigal is <contig_name>_<cds_number>
+ cds_number = gff_id.split('_')[-1]
+ cds_prodigal_id = f"{contig}_{cds_number}"
+ fh_faa.write(f">{new_id} {start}-{end}\n{protein_fa[cds_prodigal_id]}\n")
+
+
+ # Write gff and ffn
feature['attribute'] = add_new_ID_tag(feature['attribute'], new_id)
tag_name = get_tag_value(feature['attribute'], 'Name')
fh_gff.write('\t'.join(feature.values())+"\n")
-
- # feature_seq = contig_fa.fetch(contig, [int(feature['start']), int(feature['end'])])
- #
-
-
- start_zero, end_zero = int(feature['start'])-1, int(feature['end'])
if feature['strand'] == "+":
- feature_seq = contig_fa[contig][start_zero:end_zero].seq
+ feature_seq = contig_fa[contig][start-1:end].seq
else:
- # reverse complement the sequence
- feature_seq = contig_fa[contig][start_zero:end_zero].antisense
+ # minus strand: reverse complement the sequence
+ feature_seq = contig_fa[contig][start-1:end].antisense
- fh_ffn.write(f">{new_id} {tag_name}\n{feature_seq}\n")
+ fh_ffn.write(f">{new_id} {start}-{end} {tag_name}\n{feature_seq}\n")
- # if feature['feature'] == "CDS":
-
- # aa_seq = Seq(feature_seq).translate(table=translation_table, cds=True)
-
- # fh_faa.write(f'>{new_id}\n{aa_seq}\n')
-
+
+def filter_faa_file(annotation_per_contigs, faa_file, out_faa):
-# def filter_faa_file(annotation_per_contigs, faa_file, out_faa):
+ protein_fa = pyfastx.Fasta(faa_file)
-# protein_fa = pyfastx.Fasta(faa_file)
-# all_names = [set(protein_fa.keys().filter())]
+ for contig, features in annotation_per_contigs:
+
+ for feature in features:
+ new_id = f"{feature['seqname']}.{feature['feature']}_{gene_count[feature['feature']]}"
+
+ gff_id = get_tag_value(feature['attribute'], 'ID')
+ # ID in gff from prodigal is <contig_number>_<cds_number>
+ # seq name from prodigal is <contig_name>_<cds_number>
+ cds_number = gff_id.split('_')[-1]
+ cds_prodigal_id = f"{contig}_{cds_number}"
+ print(cds_prodigal_id, new_id)
+ print(protein_fa[cds_prodigal_id])
+
+
+
+
+ input()
-
-# for contig, features in annotation_per_contigs:
-# yield contig, features
+ yield contig, features
def main():
@@ -242,7 +261,7 @@ def main():
# annotation_per_contigs = filter_faa_file(annotation_per_contigs, faa_file, out_faa)
logging.info(f'Writting CDS and RNA annotations to gff file: {output_gff}.')
- writing_features_to_gff_and_ffn(annotation_per_contigs, output_gff, fna_file, out_ffn) #, out_faa)
+ writing_features_to_gff_ffn_faa(annotation_per_contigs, output_gff, fna_file, out_ffn, faa_file, out_faa)
if __name__ == '__main__':
--
GitLab
From a4466b1b9005e06dfcca963b3e21f87d2b6d5b2d Mon Sep 17 00:00:00 2001
From: Jean Mainguy <jean.mainguy@inra.fr>
Date: Thu, 20 Oct 2022 14:37:34 +0200
Subject: [PATCH 090/194] improve presentation string
---
bin/merge_annotations.py | 41 ++++++++--------------------------------
1 file changed, 8 insertions(+), 33 deletions(-)
diff --git a/bin/merge_annotations.py b/bin/merge_annotations.py
index e6a5c59..91af848 100755
--- a/bin/merge_annotations.py
+++ b/bin/merge_annotations.py
@@ -3,10 +3,11 @@
"""
Combine structural annotations made from different gff.
-In case of collapsing annotations RNA annotation are prefered to CDS annotations.
+In case of collapsing annotations, RNA annotation are prefered to CDS annotations.
+Faa file from prodigal is processed to filter out overlapping sequences.
:Example:
-python template.py -v
+merge_annotations.py -c prodigal.gff -r barrnap.gff -t trnascan_se.gff --contig_seq contigs.fasta --faa_file prodigal.faa
"""
# Metadata
@@ -37,14 +38,17 @@ def parse_arguments():
parser.add_argument('-t', '--trna', help='gff file with tRNA annotations.', required=True)
parser.add_argument('-r', '--rrna', help='gff file with rRNA annotations.', required=True)
+
+ parser.add_argument('--contig_seq', help='fasta file of contigs. Needed to extract gene sequence', required=True)
+
+ parser.add_argument('--faa_file', help='fasta file of protein sequences generated by prodigal.', required=True)
parser.add_argument('--gff_output', help='final gff file with all annotations.', default="all_annotation.gff")
parser.add_argument('--ffn_output', help='final ffn file with all annotations.', default="all_annotation.ffn")
parser.add_argument('--faa_output', help='final faa file with all CDS annotations in amino acid.', default="all_annotation.faa")
-
- parser.add_argument('--contig_seq', help='fasta file of contigs. Needed to extract gene annotations', required=True)
+
parser.add_argument("-v", "--verbose", help="increase output verbosity",
action="store_true")
@@ -191,33 +195,6 @@ def writing_features_to_gff_ffn_faa(annotations_per_contig, out_gff, fna_file, o
fh_ffn.write(f">{new_id} {start}-{end} {tag_name}\n{feature_seq}\n")
-
-def filter_faa_file(annotation_per_contigs, faa_file, out_faa):
-
- protein_fa = pyfastx.Fasta(faa_file)
-
- for contig, features in annotation_per_contigs:
-
- for feature in features:
- new_id = f"{feature['seqname']}.{feature['feature']}_{gene_count[feature['feature']]}"
-
- gff_id = get_tag_value(feature['attribute'], 'ID')
- # ID in gff from prodigal is <contig_number>_<cds_number>
- # seq name from prodigal is <contig_name>_<cds_number>
- cds_number = gff_id.split('_')[-1]
- cds_prodigal_id = f"{contig}_{cds_number}"
- print(cds_prodigal_id, new_id)
- print(protein_fa[cds_prodigal_id])
-
-
-
-
- input()
-
-
-
- yield contig, features
-
def main():
@@ -258,8 +235,6 @@ def main():
logging.info('Merge CDS and RNA annotations by contig.')
annotation_per_contigs = merging_cds_and_rna(unoverlapping_cds_per_contig, contig2rnas)
- # annotation_per_contigs = filter_faa_file(annotation_per_contigs, faa_file, out_faa)
-
logging.info(f'Writting CDS and RNA annotations to gff file: {output_gff}.')
writing_features_to_gff_ffn_faa(annotation_per_contigs, output_gff, fna_file, out_ffn, faa_file, out_faa)
--
GitLab
From 234a73a8e2552ad85539b385136cfda8c7f74971 Mon Sep 17 00:00:00 2001
From: Jean Mainguy <jean.mainguy@inra.fr>
Date: Thu, 20 Oct 2022 14:40:52 +0200
Subject: [PATCH 091/194] clean script
---
bin/merge_annotations.py | 9 +++++----
1 file changed, 5 insertions(+), 4 deletions(-)
diff --git a/bin/merge_annotations.py b/bin/merge_annotations.py
index 91af848..ac098de 100755
--- a/bin/merge_annotations.py
+++ b/bin/merge_annotations.py
@@ -212,13 +212,14 @@ def main():
rrna_file = args.rrna
cds_file = args.cds
- output_gff = args.gff_output
fna_file = args.contig_seq
+ faa_file = args.faa_file
+
+ out_gff = args.gff_output
out_ffn = args.ffn_output
out_faa = args.faa_output
- faa_file = "prodigal.faa"
logging.info('Parsing rRNA annoations.')
rrna_annotations = parse_gff_file(rrna_file, 'rRNA')
@@ -235,8 +236,8 @@ def main():
logging.info('Merge CDS and RNA annotations by contig.')
annotation_per_contigs = merging_cds_and_rna(unoverlapping_cds_per_contig, contig2rnas)
- logging.info(f'Writting CDS and RNA annotations to gff file: {output_gff}.')
- writing_features_to_gff_ffn_faa(annotation_per_contigs, output_gff, fna_file, out_ffn, faa_file, out_faa)
+ logging.info(f'Writting CDS and RNA annotations to gff file: {out_gff}.')
+ writing_features_to_gff_ffn_faa(annotation_per_contigs, out_gff, fna_file, out_ffn, faa_file, out_faa)
if __name__ == '__main__':
--
GitLab
From 489a89f2689f5b32d181383aafca4b2e0c984744 Mon Sep 17 00:00:00 2001
From: Jean Mainguy <jean.mainguy@inra.fr>
Date: Thu, 20 Oct 2022 14:45:55 +0200
Subject: [PATCH 092/194] add faa files into merging process
---
modules/merge_annotations.nf | 8 +++++---
subworkflows/04_structural_annot.nf | 2 +-
2 files changed, 6 insertions(+), 4 deletions(-)
diff --git a/modules/merge_annotations.nf b/modules/merge_annotations.nf
index 4cbbd82..12f37e8 100644
--- a/modules/merge_annotations.nf
+++ b/modules/merge_annotations.nf
@@ -2,14 +2,16 @@ process MERGE_ANNOTATIONS {
tag "${meta.id}"
input:
- tuple val(meta), file(assembly_file), file(cds_gff), file(rrna_gff), file(trna_gff)
+ tuple val(meta), file(assembly_file), file(faa_file), file(cds_gff), file(rrna_gff), file(trna_gff)
output:
+
script:
"""
- merge_annotations.py -c $cds_gff -r $rrna_gff -t $trna_gff -v --contig_seq $assembly_file \
- --ffn_output ${meta.sample}.ffn --gff_output ${meta.sample}.gff
+ merge_annotations.py -c $cds_gff -r $rrna_gff -t $trna_gff -v \
+ --contig_seq $assembly_file --faa_file $faa_file \
+ --ffn_output ${meta.sample}.ffn --gff_output ${meta.sample}.gff --faa_output ${meta.sample}.faa
"""
}
\ No newline at end of file
diff --git a/subworkflows/04_structural_annot.nf b/subworkflows/04_structural_annot.nf
index e420d55..7ce7d5e 100644
--- a/subworkflows/04_structural_annot.nf
+++ b/subworkflows/04_structural_annot.nf
@@ -13,7 +13,7 @@ workflow STEP_04_STRUCTURAL_ANNOT {
TRNASCAN_SE( assembly )
- annotations_ch = assembly.join(PRODIGAL.out.gff).join(BARRNAP.out.gff)
+ annotations_ch = assembly.join(PRODIGAL.out.faa).join(PRODIGAL.out.gff).join(BARRNAP.out.gff)
.join(TRNASCAN_SE.out.gff)
annotations_ch.view()
--
GitLab
From 6c3fd0302ad08a7593e8bb9dd6252d0e9601d3cb Mon Sep 17 00:00:00 2001
From: Jean Mainguy <jean.mainguy@inra.fr>
Date: Thu, 20 Oct 2022 17:38:04 +0200
Subject: [PATCH 093/194] Generate report to plot stat in multiqc
---
bin/merge_annotations.py | 21 +++++++++++++++++++--
main.nf | 8 ++++----
modules/merge_annotations.nf | 10 +++++++---
subworkflows/04_structural_annot.nf | 11 ++++-------
4 files changed, 34 insertions(+), 16 deletions(-)
diff --git a/bin/merge_annotations.py b/bin/merge_annotations.py
index ac098de..d18ebd0 100755
--- a/bin/merge_annotations.py
+++ b/bin/merge_annotations.py
@@ -49,6 +49,9 @@ def parse_arguments():
parser.add_argument('--faa_output', help='final faa file with all CDS annotations in amino acid.', default="all_annotation.faa")
+ parser.add_argument('--report_output', help='Prokka report like to be able to show annotations in multiqc.', default="annotation_report.txt")
+
+
parser.add_argument("-v", "--verbose", help="increase output verbosity",
action="store_true")
@@ -154,7 +157,6 @@ def writing_features_to_gff_ffn_faa(annotations_per_contig, out_gff, fna_file, o
contig_fa = pyfastx.Fasta(fna_file)
protein_fa = pyfastx.Fasta(faa_file)
-
with open(out_gff, 'w') as fh_gff, open(out_ffn, 'w') as fh_ffn, open(out_faa, 'w') as fh_faa:
fh_gff.write("##gff-version 3\n")
@@ -194,6 +196,16 @@ def writing_features_to_gff_ffn_faa(annotations_per_contig, out_gff, fna_file, o
fh_ffn.write(f">{new_id} {start}-{end} {tag_name}\n{feature_seq}\n")
+ # building prokka like report for multiqc
+ report = {"organism": "NA",
+ "contigs": len(contig_fa),
+ "bases": contig_fa.size }
+ report.update(gene_count)
+
+ return report
+
+
+
def main():
@@ -219,6 +231,7 @@ def main():
out_gff = args.gff_output
out_ffn = args.ffn_output
out_faa = args.faa_output
+ out_report = args.report_output
logging.info('Parsing rRNA annoations.')
@@ -237,7 +250,11 @@ def main():
annotation_per_contigs = merging_cds_and_rna(unoverlapping_cds_per_contig, contig2rnas)
logging.info(f'Writting CDS and RNA annotations to gff file: {out_gff}.')
- writing_features_to_gff_ffn_faa(annotation_per_contigs, out_gff, fna_file, out_ffn, faa_file, out_faa)
+ report = writing_features_to_gff_ffn_faa(annotation_per_contigs, out_gff, fna_file, out_ffn, faa_file, out_faa)
+
+ with open(out_report, "w") as fl:
+ fl.write(''.join(f"{k}: {v}\n" for k,v in report.items()))
+
if __name__ == '__main__':
diff --git a/main.nf b/main.nf
index cde0ad1..5184c2b 100644
--- a/main.nf
+++ b/main.nf
@@ -443,11 +443,11 @@ workflow {
if ( !params.stop_at_clean && !params.stop_at_assembly && !params.stop_at_filtering ) {
S04_STRUCTURAL_ANNOT ( ch_assembly )
- // ch_prokka_ffn = S04_STRUCTURAL_ANNOT.out.ffn
- // ch_prokka_faa = S04_STRUCTURAL_ANNOT.out.faa
- // ch_prokka_gff = S04_STRUCTURAL_ANNOT.out.gff
+ ch_prokka_ffn = S04_STRUCTURAL_ANNOT.out.ffn
+ ch_prokka_faa = S04_STRUCTURAL_ANNOT.out.faa
+ ch_prokka_gff = S04_STRUCTURAL_ANNOT.out.gff
// ch_prokka_fna = S04_STRUCTURAL_ANNOT.out.fna
- // ch_prokka_report = S04_STRUCTURAL_ANNOT.out.report
+ ch_prokka_report = S04_STRUCTURAL_ANNOT.out.report
// ch_contigs_and_reads = ch_prokka_fna
// .join(ch_reads, remainder: true)
diff --git a/modules/merge_annotations.nf b/modules/merge_annotations.nf
index 12f37e8..c6481f6 100644
--- a/modules/merge_annotations.nf
+++ b/modules/merge_annotations.nf
@@ -1,17 +1,21 @@
process MERGE_ANNOTATIONS {
+ publishDir "${params.outdir}/04_structural_annot/${meta.id}/", mode: 'copy'
tag "${meta.id}"
input:
tuple val(meta), file(assembly_file), file(faa_file), file(cds_gff), file(rrna_gff), file(trna_gff)
output:
-
-
+ tuple val(meta), file("${meta.id}.gff"), emit: gff
+ tuple val(meta), file("${meta.id}.ffn"), emit: ffn
+ tuple val(meta), file("${meta.id}.faa"), emit: faa
+ path "${meta.id}.txt", emit: report
script:
"""
merge_annotations.py -c $cds_gff -r $rrna_gff -t $trna_gff -v \
--contig_seq $assembly_file --faa_file $faa_file \
- --ffn_output ${meta.sample}.ffn --gff_output ${meta.sample}.gff --faa_output ${meta.sample}.faa
+ --ffn_output ${meta.id}.ffn --gff_output ${meta.id}.gff --faa_output ${meta.id}.faa \
+ --report_output ${meta.id}.txt
"""
}
\ No newline at end of file
diff --git a/subworkflows/04_structural_annot.nf b/subworkflows/04_structural_annot.nf
index 7ce7d5e..e315dbd 100644
--- a/subworkflows/04_structural_annot.nf
+++ b/subworkflows/04_structural_annot.nf
@@ -28,14 +28,11 @@ workflow STEP_04_STRUCTURAL_ANNOT {
ch_software_versions = PRODIGAL.out.v_prodigal.first().mix( BARRNAP.out.v_barrnap.first(),
TRNASCAN_SE.out.v_tRNAscan.first())
- // RENAME_CONTIGS_AND_GENES(PROKKA.out.prokka_results)
-
emit:
- // report = PROKKA.out.report
- // fna = RENAME_CONTIGS_AND_GENES.out.fna
- // ffn = RENAME_CONTIGS_AND_GENES.out.ffn
- // gff = RENAME_CONTIGS_AND_GENES.out.gff
- // faa = RENAME_CONTIGS_AND_GENES.out.faa
+ report = MERGE_ANNOTATIONS.out.report
+ ffn = MERGE_ANNOTATIONS.out.ffn
+ gff = MERGE_ANNOTATIONS.out.gff
+ faa = MERGE_ANNOTATIONS.out.faa
// prot_length = RENAME_CONTIGS_AND_GENES.out.prot_length
software_versions = ch_software_versions
}
\ No newline at end of file
--
GitLab
From 268edc62bd1f29ee8cfdfb445dd10562c108fcb6 Mon Sep 17 00:00:00 2001
From: Jean Mainguy <jean.mainguy@inra.fr>
Date: Thu, 20 Oct 2022 18:00:14 +0200
Subject: [PATCH 094/194] update multiqc config with new structural annotation
method
---
assets/hifi_multiqc_config.yaml | 92 +++++++++++++++++++++++++++++++++
assets/multiqc_config.yaml | 7 ++-
2 files changed, 98 insertions(+), 1 deletion(-)
create mode 100644 assets/hifi_multiqc_config.yaml
diff --git a/assets/hifi_multiqc_config.yaml b/assets/hifi_multiqc_config.yaml
new file mode 100644
index 0000000..c90beaa
--- /dev/null
+++ b/assets/hifi_multiqc_config.yaml
@@ -0,0 +1,92 @@
+report_comment: >
+ This report has been generated by the <a href="https://forgemia.inra.fr/genotoul-bioinfo/metagwgs" target="_blank">genotoul-bioinfo/metagwgs</a>
+ analysis pipeline. For information about how to interpret these results, please see the
+ <a href="https://forgemia.inra.fr/genotoul-bioinfo/metagwgs" target="_blank">documentation</a>.
+
+extra_fn_clean_trim:
+ - "hifi_"
+ - 'reads_'
+ - '.count_reads_on_contigs'
+ - '_scaffolds'
+ - '.txt'
+ - '.contigs'
+ - '.sort'
+ - "cleaned_"
+ - "raw_"
+ - '_kept_contigs'
+ - '.no_filter'
+ - '_kaiju_MEM_verbose'
+
+extra_fn_clean_exts:
+ - "_select_contigs_cpm"
+ - '.host_filter'
+
+module_order:
+ - fastqc:
+ name: 'FastQC (raw)'
+ path_filters_exclude:
+ - '*cleaned_*.zip'
+ - samtools:
+ name : 'Reads before host reads filter'
+ info: 'This section reports of the reads alignement against host genome.'
+ path_filters:
+ - '*.no_filter.flagstat'
+ - samtools:
+ name : 'Reads after host reads filter'
+ info: 'This section reports of the cleaned reads alignement against host genome.'
+ path_filters:
+ - '*.host_filter.flagstat'
+ - fastqc:
+ name: 'FastQC (cleaned)'
+ info: 'This section of the report shows FastQC after removing reads mapping the host genome.'
+ path_filters:
+ - '*cleaned_*.zip'
+ - kaiju
+ - quast:
+ name: 'Quast primary assembly'
+ info: 'This section of the report shows quast results after assembly.'
+ path_filters:
+ - '*quast_primary/*/report.tsv'
+ - quast:
+ name: 'Quast filtered assembly'
+ info: 'This section of the report shows quast results after assembly filtering.'
+ path_filters:
+ - '*quast_filtered/*/report.tsv'
+ - prokka:
+ name: 'Structural annotation'
+ info: 'This section of the report shows structural annotations results. CDS are predicted using Prodigal, rRNA using Barrnap and tRNA using tRNAscan-se.'
+ - featureCounts
+ - custom_content:
+ name: 'Binning results'
+ info: 'This section of the report shows quast results after binning the contigs into species genomes'
+ path_filters:
+ - '*mqc.tsv'
+
+report_section_order:
+ stats_table:
+ order: -1000
+ bins_quality:
+ order: -1010
+ bins_quality_count:
+ order: -1020
+ bins_quality_size:
+ order: -1030
+ binning_heatmap:
+ order: -1040
+ software_versions:
+ order: -1050
+
+prokka_fn_snames: True
+prokka_table: True
+
+featurecounts:
+ fn: '*.summary'
+ shared: true
+
+table_columns_visible:
+ FastQC (raw):
+ percent_duplicates: False
+ percent_gc: False
+ Structural annotation:
+ organism: False
+
diff --git a/assets/multiqc_config.yaml b/assets/multiqc_config.yaml
index fc5eb8e..06e9cdd 100644
--- a/assets/multiqc_config.yaml
+++ b/assets/multiqc_config.yaml
@@ -69,11 +69,14 @@ module_order:
path_filters:
- './final_assembly_flagstat/*'
- prokka:
+ name: 'Structural annotation'
+ info: 'This section of the report shows structural annotations results. CDS are predicted using Prodigal, rRNA using Barrnap and tRNA using tRNAscan-se.'
path_filters:
- './prokka_report/*'
- featureCounts:
path_filters:
- './featureCounts_report/*'
+ - '*.count_reads_on_contigs.flagstat'
- custom_content:
name: 'Binning results'
info: 'This section of the report shows quast results after binning the contigs into species genomes'
@@ -106,11 +109,12 @@ featurecounts:
fn: '*.summary'
shared: true
+
table_columns_visible:
FastQC (raw):
percent_duplicates: False
percent_gc: False
- prokka:
+ Structural annotation:
organism: False
Reads alignment on unfiltered assembly:
mapped_passed_pct: True
@@ -141,3 +145,4 @@ table_columns_visible:
# - hide
# show_hide_patterns:
# - ["_R1", "_R2"]
+
--
GitLab
From 92f748f4d0398302cc4fc057552e409d29677847 Mon Sep 17 00:00:00 2001
From: Jean Mainguy <jean.mainguy@inra.fr>
Date: Thu, 20 Oct 2022 23:16:21 +0200
Subject: [PATCH 095/194] rm prokka process and script
---
bin/Rename_contigs_and_genes.py | 166 --------------------------------
modules/prokka.nf | 48 ---------
2 files changed, 214 deletions(-)
delete mode 100755 bin/Rename_contigs_and_genes.py
delete mode 100644 modules/prokka.nf
diff --git a/bin/Rename_contigs_and_genes.py b/bin/Rename_contigs_and_genes.py
deleted file mode 100755
index 7d0e94d..0000000
--- a/bin/Rename_contigs_and_genes.py
+++ /dev/null
@@ -1,166 +0,0 @@
-#!/usr/bin/env python
-
-"""----------------------------------------------------------------------------------------------------------------------------------------------------------
- Script Name: Rename_contigs_and_genes.py
- Description: Rename contigs and genes in GFF, FAA and FFN
- files generated by PROKKA.
- Input files:
- GFF, FAA and FFN files produced by PROKKA.
- Created By: Celine Noirot and Joanna Fourquet
- Date: 2019-06-12
-----------------------------------------------------------------------------------------------------------------------------------------------------------
-"""
-
-# Metadata
-__author__ = 'Celine Noirot and Joanna Fourquet \
-- Plateforme bioinformatique Toulouse'
-__copyright__ = 'Copyright (C) 2019 INRA'
-__license__ = 'GNU General Public License'
-__version__ = '0.1'
-__email__ = 'support.bioinfo.genotoul@inra.fr'
-__status__ = 'dev'
-
-# Status: dev
-
-# Modules importation
-try:
- import argparse
- from BCBio import GFF
- from Bio.Seq import Seq
- from Bio.SeqRecord import SeqRecord
- from Bio.SeqFeature import SeqFeature, FeatureLocation
- import pprint
- from BCBio.GFF import GFFExaminer
- from Bio import SeqIO
-except ImportError as error:
- print(error)
- exit(1)
-
-# Manage parameters
-parser = argparse.ArgumentParser(description = \
-'Script which rename contigs and genes in GFF, FAA and FFN files generated by PROKKA.')
-parser.add_argument('-f', '--file', required = True, help = \
-'GFF file generated by PROKKA.')
-parser.add_argument('-faa', '--fastaFile', required = True, \
-help = 'Fasta of predicted sequence (aa) generated by PROKKA (FAA).')
-parser.add_argument('-ffn', '--ffnFile', required = True, \
-help = 'Fasta of predicted sequence (nuc) generated by PROKKA (FFN).')
-parser.add_argument('-fna', '--fnaFile', required = True, \
-help = 'Fasta of contigs generated by PROKKA.')
-parser.add_argument('-p', '--prefix', required = True, \
-help = 'Contig name prefix.')
-parser.add_argument('-oGFF', '--outGFFFile', required = True, \
-help = 'Name of output GFF file.')
-parser.add_argument('-oFAA', '--outFAAFile', required = True, \
-help = 'Filename of renamed predicted fasta sequences (aa).')
-parser.add_argument('-oFFN', '--outFFNFile', required = True, \
-help = 'Filename of renamed predicted fasta sequences (nuc).')
-parser.add_argument('-oFNA', '--outFNAFile', required = True, \
-help = 'Filename of renamed contig sequences (nuc).')
-parser.add_argument('-oprottable', '--outProtein', default = "protein_table.csv", \
-help = 'Filename for protein names correspondance table.')
-parser.add_argument('-oconttable', '--outContig', default = "contig_table.csv", \
-help = 'Filename for contig names correspondance table.')
-args = parser.parse_args()
-
-
-# Variable names informations:
-# prot: corresponds to proteins
-# ctg: corresponds to contigs
-
-# Variables initialization.
-prot_names = {}
-contig_renames = {}
-ctg_prefix = args.prefix
-prot_prefix = "Prot_"
-to_write = []
-# lecture fna
-#remplissage
-#contig_renames [ald_name]=newname
-#reecriture du fasta
-
-with open(args.fnaFile, "r") as fnaFile,\
- open(args.outFNAFile, "w") as outFNA_handle:
- for record in SeqIO.parse(fnaFile, "fasta"):
- try :
- old_ctg_name = record.id
- new_ctg_name = ctg_prefix + "_c" + old_ctg_name.split("_")[-1]
- contig_renames[old_ctg_name] = new_ctg_name
- record.id = contig_renames[old_ctg_name]
- record.description = record.description.replace(old_ctg_name,"")
- SeqIO.write(record, outFNA_handle, "fasta")
- except :
- print ("Warning FNA file : contig " + record.id + " discarded, no new name defined")
- pass
-
-with open(args.file) as gffFile,\
- open(args.outGFFFile, "w") as out_handle,\
- open(args.outProtein, "w") as fh_prot_table,\
- open(args.outContig, "w") as fh_cont_table,\
- open(args.outContig + ".sed", "w") as fh_cont_sed:
- for rec in GFF.parse(gffFile):
- # Access to contig id
- old_ctg_name = rec.id
- new_ctg_name = contig_renames[old_ctg_name]
- rec.id = new_ctg_name
-
- fh_cont_table.write(old_ctg_name + "\t" + new_ctg_name + "\n")
- fh_cont_sed.write("s/" + old_ctg_name + "/" + new_ctg_name + "/\n")
- # Access to features
- for f_index,feature in enumerate(rec.features):
- if(not(feature.qualifiers['source'][0].startswith("minced"))):
- #Generate correspondance
- old_prot_name = feature.qualifiers['ID'][0].replace("_gene","")
- prot_number = old_prot_name.split("_")[-1]
-
- subfeat_types = {subfeat.type for subfeat in feature.sub_features}
- assert len(subfeat_types) == 1, f'Subfeature have different types {subfeat_types}'
- subfeat_type = subfeat_types.pop()
-
-
- new_prot_name = f"{new_ctg_name}.{subfeat_type}_{prot_number}"
- prot_names[old_prot_name] = new_prot_name
- fh_prot_table.write(old_prot_name + "\t" + new_prot_name + "\n")
-
- #Initialize field of "gene" feature (the parent)
- rec.features[f_index].qualifiers["ID"] = new_prot_name + "_gene"
- rec.features[f_index].qualifiers["locus_tag"] = [new_prot_name]
-
- #Annotations (not prokka lines) are in sub_features
- for fsub_index,sub_feature in enumerate(feature.sub_features):
- # Update ID
- rec.features[f_index].sub_features[fsub_index].qualifiers["ID"] = new_prot_name
- rec.features[f_index].sub_features[fsub_index].qualifiers["Parent"] = []
- rec.features[f_index].sub_features[fsub_index].qualifiers["locus_tag"] = [new_prot_name]
- rec.features[f_index].sub_features[fsub_index].qualifiers["protein_id"] = [new_prot_name]
- to_write.append(rec)
-
- #Write only one time
- #print (to_write)
- GFF.write(to_write, out_handle)
-
-
-with open(args.fastaFile, "r") as handle,\
- open(args.outFAAFile, "w") as outFasta_handle:
- for record in SeqIO.parse(handle, "fasta"):
- try :
- old = record.id
- record.id = prot_names[record.id]
- record.description = record.description.replace(old + " ","")
- SeqIO.write(record, outFasta_handle, "fasta")
- except :
- print ("Warning FAA file : protein " + record.id + " discarded, no new name defined")
- pass
-
-
-with open(args.ffnFile, "r") as handle,\
- open(args.outFFNFile, "w") as outFFN_handle:
- for record in SeqIO.parse(handle, "fasta"):
- try :
- old = record.id
- record.id = prot_names[record.id]
- record.description = record.description.replace(old + " ","")
- SeqIO.write(record, outFFN_handle, "fasta")
- except :
- print ("Warning FFN file : protein " + record.id + " discarded, no new name defined")
- pass
diff --git a/modules/prokka.nf b/modules/prokka.nf
deleted file mode 100644
index 6fbd538..0000000
--- a/modules/prokka.nf
+++ /dev/null
@@ -1,48 +0,0 @@
-process PROKKA {
- tag "${meta.id}"
-
- input:
- tuple val(meta), file(assembly_file)
-
- output:
- tuple val(meta), path("PROKKA_${meta.id}"), emit: prokka_results
- path "PROKKA_${meta.id}/${meta.id}.txt", emit: report
- path "v_prokka.txt", emit: v_prokka
-
- script:
- """
- prokka --metagenome --noanno --rawproduct --outdir PROKKA_${meta.id} --prefix ${meta.id} ${assembly_file} --centre X --compliant --cpus ${task.cpus}
- rm PROKKA_${meta.id}/*.gbk
- gt gff3validator PROKKA_${meta.id}/${meta.id}.gff
-
- prokka -v &> v_prokka.txt
- """
-}
-
-process RENAME_CONTIGS_AND_GENES {
- tag "${meta.id}"
- publishDir "${params.outdir}/04_structural_annot", mode: 'copy'
- label 'PYTHON'
-
- input:
- tuple val(meta), path(prokka_results)
-
- output:
- tuple val(meta), path("${meta.id}.annotated.fna"), emit: fna
- tuple val(meta), path("${meta.id}.annotated.ffn"), emit: ffn
- tuple val(meta), path("${meta.id}.annotated.faa"), emit: faa
- tuple val(meta), path("${meta.id}.annotated.gff"), emit: gff
- tuple val(meta), path("${meta.id}_prot.len"), emit: prot_length
-
- script:
- """
- grep "^gnl" ${prokka_results}/${meta.id}.gff > ${meta.id}_only_gnl.gff
-
- Rename_contigs_and_genes.py -f ${meta.id}_only_gnl.gff -faa ${prokka_results}/${meta.id}.faa \
- -ffn ${prokka_results}/${meta.id}.ffn -fna ${prokka_results}/${meta.id}.fna \
- -p ${meta.id} -oGFF ${meta.id}.annotated.gff -oFAA ${meta.id}.annotated.faa \
- -oFFN ${meta.id}.annotated.ffn -oFNA ${meta.id}.annotated.fna
-
- samtools faidx ${meta.id}.annotated.faa; cut -f 1,2 ${meta.id}.annotated.faa.fai > ${meta.id}_prot.len
- """
-}
--
GitLab
From a156b1efc0b9e7b0ccf39f0db3d18a388f69c18b Mon Sep 17 00:00:00 2001
From: Jean Mainguy <jean.mainguy@inra.fr>
Date: Thu, 20 Oct 2022 23:18:32 +0200
Subject: [PATCH 096/194] rm conf of removed process
---
conf/base.config | 7 -------
conf/test.config | 7 -------
2 files changed, 14 deletions(-)
diff --git a/conf/base.config b/conf/base.config
index 607ae05..dd61115 100644
--- a/conf/base.config
+++ b/conf/base.config
@@ -58,13 +58,6 @@ process {
memory = { 8.GB * task.attempt }
cpus = 4
}
- withName: PROKKA {
- memory = { 45.GB * task.attempt }
- cpus = 8
- }
- withName: RENAME_CONTIGS_AND_GENES {
- memory = { 20.GB * task.attempt }
- }
withLabel: CD_HIT {
memory = { 50.GB * task.attempt }
cpus = 16
diff --git a/conf/test.config b/conf/test.config
index 710b959..ae50b57 100644
--- a/conf/test.config
+++ b/conf/test.config
@@ -49,13 +49,6 @@ process {
memory = { 1.GB * task.attempt }
cpus = 2
}
- withName: PROKKA {
- memory = { 1.GB * task.attempt }
- cpus = 1
- }
- withName: RENAME_CONTIGS_AND_GENES {
- memory = { 1.GB * task.attempt }
- }
withLabel: CD_HIT {
memory = { 16.GB * task.attempt }
cpus = 2
--
GitLab
From 8ac5c403aab4dcf62e4736bcfc1fb95063ec5102 Mon Sep 17 00:00:00 2001
From: Jean Mainguy <jean.mainguy@inra.fr>
Date: Thu, 20 Oct 2022 23:38:45 +0200
Subject: [PATCH 097/194] simple script to rename contigs with sample and
contig number
---
modules/rename_contigs.nf | 0
1 file changed, 0 insertions(+), 0 deletions(-)
create mode 100644 modules/rename_contigs.nf
diff --git a/modules/rename_contigs.nf b/modules/rename_contigs.nf
new file mode 100644
index 0000000..e69de29
--
GitLab
From 358893f4035ed7f786f6e32aaea2c21fdb7d838b Mon Sep 17 00:00:00 2001
From: Jean Mainguy <jean.mainguy@inra.fr>
Date: Thu, 20 Oct 2022 23:39:13 +0200
Subject: [PATCH 098/194] make script exec
---
bin/rename_contigs.py | 71 +++++++++++++++++++++++++++++++++++++++++++
1 file changed, 71 insertions(+)
create mode 100755 bin/rename_contigs.py
diff --git a/bin/rename_contigs.py b/bin/rename_contigs.py
new file mode 100755
index 0000000..0734c9c
--- /dev/null
+++ b/bin/rename_contigs.py
@@ -0,0 +1,71 @@
+#!/usr/bin/env python3
+
+"""
+Rename assembly contigs.
+
+Rename contig as <sample_name>_c<contig_number>
+
+:Example:
+rename_contigs.py -h
+"""
+
+# Metadata
+__author__ = 'Mainguy Jean - Plateforme bioinformatique Toulouse'
+__copyright__ = 'Copyright (C) 2020 INRAE'
+__license__ = 'GNU General Public License'
+__version__ = '0.1'
+__email__ = 'support.bioinfo.genotoul@inra.fr'
+__status__ = 'dev'
+
+
+from argparse import ArgumentParser, ArgumentDefaultsHelpFormatter, FileType
+import logging
+import gzip
+import csv
+from collections import defaultdict
+import pyfastx
+from Bio.Seq import Seq
+
+
+def parse_arguments():
+ """Parse script arguments."""
+ parser = ArgumentParser(description="...",
+ formatter_class=ArgumentDefaultsHelpFormatter)
+
+ parser.add_argument('-s', '--sample', help='Sample name used to rename contigs.', required=True)
+
+ parser.add_argument('-i', '--fna_file', help='Original fasta file of contigs.', required=True)
+
+ parser.add_argument('-o', '--out_fna', help='Output fasta file with renamed contigs.', required=True)
+
+ parser.add_argument("-v", "--verbose", help="increase output verbosity",
+ action="store_true")
+
+ args = parser.parse_args()
+ return args
+
+
+
+def main():
+
+ args = parse_arguments()
+
+ if args.verbose:
+ logging.basicConfig(format="%(levelname)s: %(message)s", level=logging.DEBUG)
+ logging.info('Mode verbose ON')
+
+ else:
+ logging.basicConfig(format="%(levelname)s: %(message)s")
+
+ sample = args.sample
+ fna_file = args.fna_file
+ out_fna = args.out_fna
+
+
+ logging.info(f'Writting renamed fasta file in {out_fna}')
+ with open(out_fna, "w") as fh_fna:
+ for i, (_, seq) in enumerate(pyfastx.Fasta(fna_file, build_index=False)):
+ fh_fna.write(f'>{sample}_c{i+1}\n{seq}\n')
+
+if __name__ == '__main__':
+ main()
\ No newline at end of file
--
GitLab
From 28d6f34f96d43ac42533df61fb6be28c7b281664 Mon Sep 17 00:00:00 2001
From: Jean Mainguy <jean.mainguy@inra.fr>
Date: Thu, 20 Oct 2022 23:42:47 +0200
Subject: [PATCH 099/194] call RENAME_CONTIGS process
---
modules/assembly.nf | 17 +++++++++++++++++
1 file changed, 17 insertions(+)
diff --git a/modules/assembly.nf b/modules/assembly.nf
index b8f9d35..41db2c5 100644
--- a/modules/assembly.nf
+++ b/modules/assembly.nf
@@ -107,3 +107,20 @@ process METAFLYE {
"""
}
+process RENAME_CONTIGS {
+ tag "${meta.id}"
+ publishDir "${params.outdir}/02_assembly/", mode: 'copy'
+ label 'PYTHON'
+
+ input:
+ tuple val(meta), path(assembly)
+
+ output:
+ tuple val(meta), path("${meta.id}.fna"), emit: fna
+
+ script:
+ """
+ rename_contigs.py --sample ${meta.id} --fna_file $assembly --out_fna ${meta.id}.fna -v
+
+ """
+}
\ No newline at end of file
--
GitLab
From 752a97b7d82d56250603cb50e6b7c01e12cfc4cc Mon Sep 17 00:00:00 2001
From: Jean Mainguy <jean.mainguy@inra.fr>
Date: Thu, 20 Oct 2022 23:50:28 +0200
Subject: [PATCH 100/194] call RENAME_CONTIGS in 02_assembly
---
modules/rename_contigs.nf | 0
subworkflows/02_assembly.nf | 9 +++++----
2 files changed, 5 insertions(+), 4 deletions(-)
delete mode 100644 modules/rename_contigs.nf
diff --git a/modules/rename_contigs.nf b/modules/rename_contigs.nf
deleted file mode 100644
index e69de29..0000000
diff --git a/subworkflows/02_assembly.nf b/subworkflows/02_assembly.nf
index 8411728..0101234 100644
--- a/subworkflows/02_assembly.nf
+++ b/subworkflows/02_assembly.nf
@@ -1,4 +1,4 @@
-include { METASPADES; MEGAHIT; HIFIASM_META; METAFLYE } from '../modules/assembly'
+include { METASPADES; MEGAHIT; HIFIASM_META; METAFLYE; RENAME_CONTIGS } from '../modules/assembly'
include { QUAST as ASSEMBLY_QUAST} from '../modules/metaquast'
include { READS_DEDUPLICATION } from '../modules/reads_deduplication'
include { MINIMAP2 } from '../modules/read_alignment'
@@ -47,7 +47,8 @@ workflow STEP_02_ASSEMBLY {
}
}
- ASSEMBLY_QUAST( ch_assembly,"02_assembly/quast_primary")
+ ch_assembly_renamed = RENAME_CONTIGS(ch_assembly)
+ ASSEMBLY_QUAST( ch_assembly_renamed,"02_assembly/quast_primary")
ch_assembly_report = ASSEMBLY_QUAST.out.report
ch_quast_v = ASSEMBLY_QUAST.out.v_quast
@@ -57,7 +58,7 @@ workflow STEP_02_ASSEMBLY {
ch_flagstat = Channel.empty()
ch_reads = reads
- ch_contigs_and_reads = ch_assembly.join(ch_reads, remainder: true)
+ ch_contigs_and_reads = ch_assembly_renamed.join(ch_reads, remainder: true)
if ( params.type.toUpperCase() == "SR" ) {
READS_DEDUPLICATION ( ch_contigs_and_reads )
ch_dedup = READS_DEDUPLICATION.out.dedup
@@ -88,7 +89,7 @@ workflow STEP_02_ASSEMBLY {
emit:
- assembly = ch_assembly
+ assembly = ch_assembly_renamed
dedup = ch_dedup
idxstats = ch_idxstats
flagstat = ch_flagstat
--
GitLab
From 477d5fac8ed6ce58102d0a40ceadd923c4a3c681 Mon Sep 17 00:00:00 2001
From: Jean Mainguy <jean.mainguy@inra.fr>
Date: Mon, 24 Oct 2022 12:21:20 +0200
Subject: [PATCH 101/194] simplify quast report channel
---
main.nf | 13 ++++++++-----
modules/metaquast.nf | 2 +-
2 files changed, 9 insertions(+), 6 deletions(-)
diff --git a/main.nf b/main.nf
index 5184c2b..fd9fffe 100644
--- a/main.nf
+++ b/main.nf
@@ -404,12 +404,16 @@ workflow {
/////////////////////
S02_ASSEMBLY ( ch_preprocessed_reads, ch_assembly, has_assembly, assembly_tool )
+
ch_assembly = S02_ASSEMBLY.out.assembly
- ch_reads = S02_ASSEMBLY.out.dedup
+ ch_reads = S02_ASSEMBLY.out.reads
+
+
ch_idxstats = S02_ASSEMBLY.out.idxstats
+
ch_unfilter_assembly_flagstat_report = S02_ASSEMBLY.out.flagstat
- ch_quast = S02_ASSEMBLY.out.assembly_report
- ch_quast_before_filter_report = ch_quast.map{ meta, quast_report -> quast_report }
+
+ ch_quast_before_filter_report = S02_ASSEMBLY.out.assembly_report
ch_software_versions_S02 = S02_ASSEMBLY.out.software_versions
}
@@ -429,8 +433,7 @@ workflow {
ch_min_contigs_cpm
)
ch_assembly = S03_FILTERING.out.selected
- ch_quast = S03_FILTERING.out.report
- ch_quast_after_filter_report = ch_quast.map{ meta, quast_report -> quast_report }
+ ch_quast_after_filter_report = S03_FILTERING.out.report
}
diff --git a/modules/metaquast.nf b/modules/metaquast.nf
index dbaa508..dee90d5 100644
--- a/modules/metaquast.nf
+++ b/modules/metaquast.nf
@@ -9,7 +9,7 @@ process QUAST {
output:
path "${outdir}/${meta.id}/*", emit: all
- tuple val(meta), path ("${outdir}/${meta.id}/report.tsv"), emit: report
+ path "${outdir}/${meta.id}/report.tsv", emit: report
path "v_quast.txt", emit: v_quast
script:
--
GitLab
From 3be023ffc1214fd90b7ffb2bb188e8567d1cdb66 Mon Sep 17 00:00:00 2001
From: Jean Mainguy <jean.mainguy@inra.fr>
Date: Mon, 24 Oct 2022 14:30:19 +0200
Subject: [PATCH 102/194] clean subworkflow STEP_04_STRUCTURAL_ANNOT
---
subworkflows/04_structural_annot.nf | 6 ------
1 file changed, 6 deletions(-)
diff --git a/subworkflows/04_structural_annot.nf b/subworkflows/04_structural_annot.nf
index e315dbd..edac18e 100644
--- a/subworkflows/04_structural_annot.nf
+++ b/subworkflows/04_structural_annot.nf
@@ -12,19 +12,13 @@ workflow STEP_04_STRUCTURAL_ANNOT {
BARRNAP( assembly )
TRNASCAN_SE( assembly )
-
annotations_ch = assembly.join(PRODIGAL.out.faa).join(PRODIGAL.out.gff).join(BARRNAP.out.gff)
.join(TRNASCAN_SE.out.gff)
- annotations_ch.view()
-
-
MERGE_ANNOTATIONS(annotations_ch)
-
ch_software_versions = PRODIGAL.out.v_prodigal.first()
-
ch_software_versions = PRODIGAL.out.v_prodigal.first().mix( BARRNAP.out.v_barrnap.first(),
TRNASCAN_SE.out.v_tRNAscan.first())
--
GitLab
From 6b29a57c4bbf8e98756781291852450bcdf5e056 Mon Sep 17 00:00:00 2001
From: Jean Mainguy <jean.mainguy@inra.fr>
Date: Mon, 24 Oct 2022 14:34:12 +0200
Subject: [PATCH 103/194] reorganise deduplication process
---
main.nf | 2 +-
modules/reads_deduplication.nf | 43 ++++++++++++++++++++--------------
subworkflows/02_assembly.nf | 20 ++++++++++------
3 files changed, 39 insertions(+), 26 deletions(-)
diff --git a/main.nf b/main.nf
index fd9fffe..bba6ea7 100644
--- a/main.nf
+++ b/main.nf
@@ -407,7 +407,7 @@ workflow {
ch_assembly = S02_ASSEMBLY.out.assembly
ch_reads = S02_ASSEMBLY.out.reads
-
+ ch_bam = S02_ASSEMBLY.out.bam
ch_idxstats = S02_ASSEMBLY.out.idxstats
diff --git a/modules/reads_deduplication.nf b/modules/reads_deduplication.nf
index b64df7c..4731f9b 100644
--- a/modules/reads_deduplication.nf
+++ b/modules/reads_deduplication.nf
@@ -1,38 +1,45 @@
process READS_DEDUPLICATION {
tag "${meta.id}"
- publishDir "${params.outdir}/02_assembly", mode: 'copy', pattern: '*.fastq.gz'
- publishDir "${params.outdir}/02_assembly/logs", mode: 'copy', pattern: '*.idxstats'
- publishDir "${params.outdir}/02_assembly/logs", mode: 'copy', pattern: '*.flagstat'
+ publishDir "${params.outdir}/02_assembly/deduplicated_reads/", mode: 'copy', pattern: '*.fastq.gz'
+ publishDir "${params.outdir}/02_assembly/${meta.id}/", mode: 'copy', pattern: '*.fastq.gz'
input:
tuple val(meta), path(assembly), path(reads)
output:
- tuple val(meta), path("${meta.id}*_dedup.fastq.gz"), emit: dedup
- tuple val(meta), path("${meta.id}.count_reads_on_contigs.idxstats"), emit: idxstats
+ tuple val(meta), path("${meta.id}*_dedup.fastq.gz"), emit: dedup_reads
+ tuple val(meta), path("${meta.id}.bam"), path("${meta.id}.bam.bai"), emit: bam
+
path "${meta.id}_singletons.fastq.gz", emit: singletons
- path "${meta.id}.count_reads_on_contigs.flagstat", emit: flagstat
+
path "v_bwa.txt", emit: v_bwa_mem2
path "v_samtools.txt", emit: v_samtools
script:
"""
- mkdir logs
+ # Align reads against assembly
bwa-mem2 index ${assembly} -p ${assembly}
- bwa-mem2 mem ${assembly} ${reads[0]} ${reads[1]} | samtools view -bS - | samtools sort -n -o ${meta.id}.sort.bam -
- samtools fixmate -m ${meta.id}.sort.bam ${meta.id}.fixmate.bam
- samtools sort -o ${meta.id}.fixmate.positionsort.bam ${meta.id}.fixmate.bam
- samtools markdup -r -S -s -f ${meta.id}.stats ${meta.id}.fixmate.positionsort.bam ${meta.id}.filtered.bam
- samtools index ${meta.id}.filtered.bam
- samtools idxstats ${meta.id}.filtered.bam > ${meta.id}.count_reads_on_contigs.idxstats
- samtools flagstat ${meta.id}.filtered.bam > ${meta.id}.count_reads_on_contigs.flagstat
- samtools sort -n -o ${meta.id}.filtered.sort.bam ${meta.id}.filtered.bam
- samtools fastq -N -s ${meta.id}_singletons.fastq.gz -1 ${meta.id}_R1_dedup.fastq.gz -2 ${meta.id}_R2_dedup.fastq.gz ${meta.id}.filtered.sort.bam
+ bwa-mem2 mem -t ${task.cpus} ${assembly} ${reads[0]} ${reads[1]} | samtools view -@ ${task.cpus} -bS - | samtools sort -@ ${task.cpus} -n -o ${meta.id}.sort.bam -
+
+ # Identify and removed duplicated reads from the bam file
+ samtools fixmate -@ ${task.cpus} -m ${meta.id}.sort.bam ${meta.id}.fixmate.bam
+ samtools sort -@ ${task.cpus} -o ${meta.id}.fixmate.positionsort.bam ${meta.id}.fixmate.bam
+ samtools markdup -@ ${task.cpus} -r -S -s -f ${meta.id}.stats ${meta.id}.fixmate.positionsort.bam ${meta.id}.filtered.bam
+
+ # final bam file without duplicated reads
+ samtools sort -@ ${task.cpus} -o ${meta.id}.bam ${meta.id}.filtered.bam
+ samtools index -@ ${task.cpus} ${meta.id}.bam
+
+ # Get deduplicated reads
+ samtools sort -@ ${task.cpus} -n -o ${meta.id}.filtered.n_sorted.bam ${meta.id}.bam
+ samtools fastq -@ ${task.cpus} -N -s ${meta.id}_singletons.fastq.gz -1 ${meta.id}_R1_dedup.fastq.gz -2 ${meta.id}_R2_dedup.fastq.gz ${meta.id}.filtered.n_sorted.bam
+
+ # clean directory
rm ${meta.id}.sort.bam
rm ${meta.id}.fixmate.bam
rm ${meta.id}.fixmate.positionsort.bam
- rm ${meta.id}.filtered.bam
- rm ${meta.id}.filtered.sort.bam
+ rm ${meta.id}.filtered.n_sorted.bam
+ rm ${meta.id}.filtered.bam
bwa-mem2 version > v_bwa.txt
diff --git a/subworkflows/02_assembly.nf b/subworkflows/02_assembly.nf
index 0101234..6f6141e 100644
--- a/subworkflows/02_assembly.nf
+++ b/subworkflows/02_assembly.nf
@@ -52,26 +52,31 @@ workflow STEP_02_ASSEMBLY {
ch_assembly_report = ASSEMBLY_QUAST.out.report
ch_quast_v = ASSEMBLY_QUAST.out.v_quast
- ch_dedup = Channel.empty()
ch_idxstats = Channel.empty()
- ch_dedup_report = Channel.empty()
ch_flagstat = Channel.empty()
ch_reads = reads
ch_contigs_and_reads = ch_assembly_renamed.join(ch_reads, remainder: true)
if ( params.type.toUpperCase() == "SR" ) {
+
READS_DEDUPLICATION ( ch_contigs_and_reads )
- ch_dedup = READS_DEDUPLICATION.out.dedup
- ch_idxstats = READS_DEDUPLICATION.out.idxstats
- ch_flagstat = READS_DEDUPLICATION.out.flagstat
+
+ ch_reads = READS_DEDUPLICATION.out.dedup_reads
+ ch_bam = READS_DEDUPLICATION.out.bam
+
+ SAMTOOLS(ch_bam, "02_assembly")
+ ch_idxstats = SAMTOOLS.out.sam_idxstat
+ ch_flagstat = SAMTOOLS.out.sam_flagstat
ch_bwa_mem_v = READS_DEDUPLICATION.out.v_bwa_mem2
ch_samtools_v = READS_DEDUPLICATION.out.v_samtools
+
} else {
- ch_dedup = ch_reads
+
if (!params.skip_filtering) {
MINIMAP2(ch_contigs_and_reads,"02_assembly")
ch_bam = MINIMAP2.out.bam
+
SAMTOOLS(ch_bam, "02_assembly")
ch_idxstats = SAMTOOLS.out.sam_idxstat
ch_flagstat = SAMTOOLS.out.sam_flagstat
@@ -90,7 +95,8 @@ workflow STEP_02_ASSEMBLY {
emit:
assembly = ch_assembly_renamed
- dedup = ch_dedup
+ reads = ch_reads
+ bam = ch_bam
idxstats = ch_idxstats
flagstat = ch_flagstat
assembly_report = ch_assembly_report
--
GitLab
From a24c26d4b4c18fcfc8c15451e05051c9c93eaf82 Mon Sep 17 00:00:00 2001
From: Jean Mainguy <jean.mainguy@inra.fr>
Date: Mon, 24 Oct 2022 14:35:00 +0200
Subject: [PATCH 104/194] correct typo in multiqc_config
---
assets/multiqc_config.yaml | 2 +-
1 file changed, 1 insertion(+), 1 deletion(-)
diff --git a/assets/multiqc_config.yaml b/assets/multiqc_config.yaml
index 06e9cdd..c24c305 100644
--- a/assets/multiqc_config.yaml
+++ b/assets/multiqc_config.yaml
@@ -75,7 +75,7 @@ module_order:
- './prokka_report/*'
- featureCounts:
path_filters:
- - './featureCounts_report/*'
+ - './featureCounts_report/*'
- '*.count_reads_on_contigs.flagstat'
- custom_content:
name: 'Binning results'
--
GitLab
From 6959b9bd702a1e6800ad2805ced05f36e83d72ec Mon Sep 17 00:00:00 2001
From: Jean Mainguy <jean.mainguy@inra.fr>
Date: Mon, 24 Oct 2022 14:37:21 +0200
Subject: [PATCH 105/194] rm tax plot made from prot length
---
bin/aln2taxaffi.py | 15 ++++++++-------
main.nf | 4 +---
subworkflows/07_taxonomic_affi.nf | 2 --
3 files changed, 9 insertions(+), 12 deletions(-)
diff --git a/bin/aln2taxaffi.py b/bin/aln2taxaffi.py
index 5c85116..241d836 100755
--- a/bin/aln2taxaffi.py
+++ b/bin/aln2taxaffi.py
@@ -597,13 +597,14 @@ def main():
logging.debug(contig_affi_line)
-
- with open(query_length_file) as fl:
- nb_total_prot = len([line for line in fl])
-
- nb_prot_annotated = len(matches)
- plot_taxonomic_assignment(
- output_name, count_rank_affiliation_protein, count_rank_affiliation_contig, nb_total_prot, nb_prot_annotated, nb_prot_assigned)
+ if query_length_file:
+ logging.debug("Plot taxonomic affiliation using protein lengths.")
+ with open(query_length_file) as fl:
+ nb_total_prot = len([line for line in fl])
+
+ nb_prot_annotated = len(matches)
+ plot_taxonomic_assignment(
+ output_name, count_rank_affiliation_protein, count_rank_affiliation_contig, nb_total_prot, nb_prot_annotated, nb_prot_assigned)
if args.write_top_taxons:
top_taxon_columns = ['contig', ] + main_ranks
diff --git a/main.nf b/main.nf
index bba6ea7..75c9f7b 100644
--- a/main.nf
+++ b/main.nf
@@ -442,7 +442,6 @@ workflow {
ch_prokka_faa = Channel.empty()
ch_prokka_gff = Channel.empty()
ch_prokka_fna = Channel.empty()
- ch_prot_length = Channel.empty()
if ( !params.stop_at_clean && !params.stop_at_assembly && !params.stop_at_filtering ) {
S04_STRUCTURAL_ANNOT ( ch_assembly )
@@ -454,7 +453,6 @@ workflow {
// ch_contigs_and_reads = ch_prokka_fna
// .join(ch_reads, remainder: true)
- // ch_prot_length = S04_STRUCTURAL_ANNOT.out.prot_length
ch_software_versions_S04 = S04_STRUCTURAL_ANNOT.out.software_versions
// exit 0, 'Exit now... S04_STRUCTURAL_ANNOT being constructed '
@@ -487,7 +485,7 @@ workflow {
}
if ( !params.stop_at_clean && !params.stop_at_assembly && !params.stop_at_filtering && !params.stop_at_structural_annot && !params.skip_taxo_affi ) {
- S07_TAXO_AFFI ( ch_taxonomy, ch_m8, ch_sam_coverage, ch_prot_length)
+ S07_TAXO_AFFI ( ch_taxonomy, ch_m8, ch_sam_coverage)
ch_software_versions_S07 = S07_TAXO_AFFI.out.software_versions
}
diff --git a/subworkflows/07_taxonomic_affi.nf b/subworkflows/07_taxonomic_affi.nf
index d1042c0..8eb2640 100644
--- a/subworkflows/07_taxonomic_affi.nf
+++ b/subworkflows/07_taxonomic_affi.nf
@@ -7,10 +7,8 @@ workflow STEP_07_TAXO_AFFI {
taxonomy
diamond_result // channel: [ val(meta), path(diamond_file) ]
sam_coverage // channel: [ val(meta), path(samtools coverage) ]
- prot_length // channel: [ val(meta), path(prot_length) ]
main:
ch_assign_taxo_input = diamond_result.join(sam_coverage, remainder: true)
- .join(prot_length, remainder: true)
ASSIGN_TAXONOMY ( taxonomy, ch_assign_taxo_input )
--
GitLab
From ce2b34a68b8ad729937075c10b6f53eceb2fe8da Mon Sep 17 00:00:00 2001
From: Jean Mainguy <jean.mainguy@inra.fr>
Date: Mon, 17 Oct 2022 15:51:34 +0200
Subject: [PATCH 106/194] Add barrnap module see issue #163
---
modules/barnap.nf | 18 ++++++++++++++++++
1 file changed, 18 insertions(+)
create mode 100644 modules/barnap.nf
diff --git a/modules/barnap.nf b/modules/barnap.nf
new file mode 100644
index 0000000..0b88b76
--- /dev/null
+++ b/modules/barnap.nf
@@ -0,0 +1,18 @@
+process PRODIGAL {
+ tag "${meta.id}"
+
+ input:
+ tuple val(meta), file(assembly_file)
+
+ output:
+ tuple val(meta), path("barrnap.gff"), emit: barrnap_gff
+ path "v_barrnap.txt", emit: v_barrnap
+
+ script:
+ """
+ barrnap -o barrnap.gff --threads ${task.cpus} ${assembly_file}
+
+ barrnap --version 2> v_barrnap.txt
+
+ """
+}
--
GitLab
From 1babda4552acea64b0bb7a5a65b205c03cd818fe Mon Sep 17 00:00:00 2001
From: Jean Mainguy <jean.mainguy@inra.fr>
Date: Mon, 17 Oct 2022 15:55:34 +0200
Subject: [PATCH 107/194] fix module name
---
modules/barnap.nf | 2 +-
1 file changed, 1 insertion(+), 1 deletion(-)
diff --git a/modules/barnap.nf b/modules/barnap.nf
index 0b88b76..302ab6d 100644
--- a/modules/barnap.nf
+++ b/modules/barnap.nf
@@ -1,4 +1,4 @@
-process PRODIGAL {
+process BARRNAP {
tag "${meta.id}"
input:
--
GitLab
From 151754bd209f6a99c0f6dbb359cc09e4f8ff8c32 Mon Sep 17 00:00:00 2001
From: Jean Mainguy <jean.mainguy@inra.fr>
Date: Mon, 17 Oct 2022 16:18:15 +0200
Subject: [PATCH 108/194] fix typo in barrnap name module
---
modules/barnap.nf | 18 ------------------
1 file changed, 18 deletions(-)
delete mode 100644 modules/barnap.nf
diff --git a/modules/barnap.nf b/modules/barnap.nf
deleted file mode 100644
index 302ab6d..0000000
--- a/modules/barnap.nf
+++ /dev/null
@@ -1,18 +0,0 @@
-process BARRNAP {
- tag "${meta.id}"
-
- input:
- tuple val(meta), file(assembly_file)
-
- output:
- tuple val(meta), path("barrnap.gff"), emit: barrnap_gff
- path "v_barrnap.txt", emit: v_barrnap
-
- script:
- """
- barrnap -o barrnap.gff --threads ${task.cpus} ${assembly_file}
-
- barrnap --version 2> v_barrnap.txt
-
- """
-}
--
GitLab
From 8abe7cc667af0f1a18613c2abd22bbb59b47b080 Mon Sep 17 00:00:00 2001
From: Jean Mainguy <jean.mainguy@inra.fr>
Date: Wed, 19 Oct 2022 18:03:14 +0200
Subject: [PATCH 109/194] Add process and script to merge gff annotations issue
#163
---
bin/merge_annotations.py | 1 -
1 file changed, 1 deletion(-)
diff --git a/bin/merge_annotations.py b/bin/merge_annotations.py
index d18ebd0..8c0b3c1 100755
--- a/bin/merge_annotations.py
+++ b/bin/merge_annotations.py
@@ -126,7 +126,6 @@ def remove_overlapping_cds(cds_file, contig2rnas):
yield current_contig, contig_annotations
def merging_cds_and_rna(cds_per_contig, contig2rnas):
-
contig_processed = []
for contig, cds_features in cds_per_contig:
contig_processed.append(contig)
--
GitLab
From 98ce3bb83f1bef6ea86fe0bd686023c6ecb79cb0 Mon Sep 17 00:00:00 2001
From: Jean Mainguy <jean.mainguy@inra.fr>
Date: Wed, 19 Oct 2022 19:01:45 +0200
Subject: [PATCH 110/194] add ffn output on top of gff output
---
bin/merge_annotations.py | 4 ++++
1 file changed, 4 insertions(+)
diff --git a/bin/merge_annotations.py b/bin/merge_annotations.py
index 8c0b3c1..07e262b 100755
--- a/bin/merge_annotations.py
+++ b/bin/merge_annotations.py
@@ -233,6 +233,9 @@ def main():
out_report = args.report_output
+ fna_file = args.contig_seq
+ out_ffn = args.ffn_output
+
logging.info('Parsing rRNA annoations.')
rrna_annotations = parse_gff_file(rrna_file, 'rRNA')
@@ -256,5 +259,6 @@ def main():
+
if __name__ == '__main__':
main()
\ No newline at end of file
--
GitLab
From f85abbb97106324d34a44a8c26b8d939b4ab6ee9 Mon Sep 17 00:00:00 2001
From: Jean Mainguy <jean.mainguy@inra.fr>
Date: Thu, 20 Oct 2022 13:37:32 +0200
Subject: [PATCH 111/194] fix strand and zero base position when extracting seq
---
bin/merge_annotations.py | 35 +++++++++++++++++++++++++++++++++++
1 file changed, 35 insertions(+)
diff --git a/bin/merge_annotations.py b/bin/merge_annotations.py
index 07e262b..4353fa7 100755
--- a/bin/merge_annotations.py
+++ b/bin/merge_annotations.py
@@ -126,6 +126,7 @@ def remove_overlapping_cds(cds_file, contig2rnas):
yield current_contig, contig_annotations
def merging_cds_and_rna(cds_per_contig, contig2rnas):
+
contig_processed = []
for contig, cds_features in cds_per_contig:
contig_processed.append(contig)
@@ -205,6 +206,34 @@ def writing_features_to_gff_ffn_faa(annotations_per_contig, out_gff, fna_file, o
+ start_zero, end_zero = int(feature['start'])-1, int(feature['end'])
+
+ if feature['strand'] == "+":
+ feature_seq = contig_fa[contig][start_zero:end_zero].seq
+ else:
+ # reverse complement the sequence
+ feature_seq = contig_fa[contig][start_zero:end_zero].antisense
+
+ fh_ffn.write(f">{new_id} {tag_name}\n{feature_seq}\n")
+
+
+ # if feature['feature'] == "CDS":
+
+ # aa_seq = Seq(feature_seq).translate(table=translation_table, cds=True)
+
+ # fh_faa.write(f'>{new_id}\n{aa_seq}\n')
+
+
+# def filter_faa_file(annotation_per_contigs, faa_file, out_faa):
+
+# protein_fa = pyfastx.Fasta(faa_file)
+# all_names = [set(protein_fa.keys().filter())]
+
+
+# for contig, features in annotation_per_contigs:
+
+
+# yield contig, features
def main():
@@ -235,6 +264,9 @@ def main():
fna_file = args.contig_seq
out_ffn = args.ffn_output
+ out_faa = args.faa_output
+
+ faa_file = "prodigal.faa"
logging.info('Parsing rRNA annoations.')
rrna_annotations = parse_gff_file(rrna_file, 'rRNA')
@@ -259,6 +291,9 @@ def main():
+ logging.info(f'Writting CDS and RNA annotations to gff file: {output_gff}.')
+ writing_features_to_gff_and_ffn(annotation_per_contigs, output_gff, fna_file, out_ffn) #, out_faa)
+
if __name__ == '__main__':
main()
\ No newline at end of file
--
GitLab
From 6de5436e9abfe55f637fa11e0f49b60c42c4b5ac Mon Sep 17 00:00:00 2001
From: Jean Mainguy <jean.mainguy@inra.fr>
Date: Thu, 20 Oct 2022 14:35:49 +0200
Subject: [PATCH 112/194] add faa file filtering from prodigal
---
bin/merge_annotations.py | 43 ++++++++++++++++++++++++----------------
1 file changed, 26 insertions(+), 17 deletions(-)
diff --git a/bin/merge_annotations.py b/bin/merge_annotations.py
index 4353fa7..edc12b6 100755
--- a/bin/merge_annotations.py
+++ b/bin/merge_annotations.py
@@ -209,31 +209,40 @@ def writing_features_to_gff_ffn_faa(annotations_per_contig, out_gff, fna_file, o
start_zero, end_zero = int(feature['start'])-1, int(feature['end'])
if feature['strand'] == "+":
- feature_seq = contig_fa[contig][start_zero:end_zero].seq
+ feature_seq = contig_fa[contig][start-1:end].seq
else:
- # reverse complement the sequence
- feature_seq = contig_fa[contig][start_zero:end_zero].antisense
-
- fh_ffn.write(f">{new_id} {tag_name}\n{feature_seq}\n")
+ # minus strand: reverse complement the sequence
+ feature_seq = contig_fa[contig][start-1:end].antisense
+ fh_ffn.write(f">{new_id} {start}-{end} {tag_name}\n{feature_seq}\n")
- # if feature['feature'] == "CDS":
-
- # aa_seq = Seq(feature_seq).translate(table=translation_table, cds=True)
-
- # fh_faa.write(f'>{new_id}\n{aa_seq}\n')
+
+def filter_faa_file(annotation_per_contigs, faa_file, out_faa):
-# def filter_faa_file(annotation_per_contigs, faa_file, out_faa):
+ protein_fa = pyfastx.Fasta(faa_file)
-# protein_fa = pyfastx.Fasta(faa_file)
-# all_names = [set(protein_fa.keys().filter())]
+ for contig, features in annotation_per_contigs:
+
+ for feature in features:
+ new_id = f"{feature['seqname']}.{feature['feature']}_{gene_count[feature['feature']]}"
+
+ gff_id = get_tag_value(feature['attribute'], 'ID')
+ # ID in gff from prodigal is <contig_number>_<cds_number>
+ # seq name from prodigal is <contig_name>_<cds_number>
+ cds_number = gff_id.split('_')[-1]
+ cds_prodigal_id = f"{contig}_{cds_number}"
+ print(cds_prodigal_id, new_id)
+ print(protein_fa[cds_prodigal_id])
+
+
+
+
+ input()
-
-# for contig, features in annotation_per_contigs:
-# yield contig, features
+ yield contig, features
def main():
@@ -292,7 +301,7 @@ def main():
logging.info(f'Writting CDS and RNA annotations to gff file: {output_gff}.')
- writing_features_to_gff_and_ffn(annotation_per_contigs, output_gff, fna_file, out_ffn) #, out_faa)
+ writing_features_to_gff_ffn_faa(annotation_per_contigs, output_gff, fna_file, out_ffn, faa_file, out_faa)
if __name__ == '__main__':
--
GitLab
From 9826c73ba81e5c841028d7ad8904ae7ed89a4e17 Mon Sep 17 00:00:00 2001
From: Jean Mainguy <jean.mainguy@inra.fr>
Date: Thu, 20 Oct 2022 14:37:34 +0200
Subject: [PATCH 113/194] improve presentation string
---
bin/merge_annotations.py | 27 ---------------------------
1 file changed, 27 deletions(-)
diff --git a/bin/merge_annotations.py b/bin/merge_annotations.py
index edc12b6..2c4dbc9 100755
--- a/bin/merge_annotations.py
+++ b/bin/merge_annotations.py
@@ -217,33 +217,6 @@ def writing_features_to_gff_ffn_faa(annotations_per_contig, out_gff, fna_file, o
fh_ffn.write(f">{new_id} {start}-{end} {tag_name}\n{feature_seq}\n")
-
-def filter_faa_file(annotation_per_contigs, faa_file, out_faa):
-
- protein_fa = pyfastx.Fasta(faa_file)
-
- for contig, features in annotation_per_contigs:
-
- for feature in features:
- new_id = f"{feature['seqname']}.{feature['feature']}_{gene_count[feature['feature']]}"
-
- gff_id = get_tag_value(feature['attribute'], 'ID')
- # ID in gff from prodigal is <contig_number>_<cds_number>
- # seq name from prodigal is <contig_name>_<cds_number>
- cds_number = gff_id.split('_')[-1]
- cds_prodigal_id = f"{contig}_{cds_number}"
- print(cds_prodigal_id, new_id)
- print(protein_fa[cds_prodigal_id])
-
-
-
-
- input()
-
-
-
- yield contig, features
-
def main():
--
GitLab
From 7017a0ea818753dadc3a02a083b7dc20153d682b Mon Sep 17 00:00:00 2001
From: Jean Mainguy <jean.mainguy@inra.fr>
Date: Thu, 20 Oct 2022 14:40:52 +0200
Subject: [PATCH 114/194] clean script
---
bin/merge_annotations.py | 4 +++-
1 file changed, 3 insertions(+), 1 deletion(-)
diff --git a/bin/merge_annotations.py b/bin/merge_annotations.py
index 2c4dbc9..dbf58db 100755
--- a/bin/merge_annotations.py
+++ b/bin/merge_annotations.py
@@ -245,10 +245,12 @@ def main():
fna_file = args.contig_seq
+ faa_file = args.faa_file
+
+ out_gff = args.gff_output
out_ffn = args.ffn_output
out_faa = args.faa_output
- faa_file = "prodigal.faa"
logging.info('Parsing rRNA annoations.')
rrna_annotations = parse_gff_file(rrna_file, 'rRNA')
--
GitLab
From 9fd2ebcc134a7ce104f6da570becfdbadbaa898c Mon Sep 17 00:00:00 2001
From: Jean Mainguy <jean.mainguy@inra.fr>
Date: Thu, 20 Oct 2022 18:00:14 +0200
Subject: [PATCH 115/194] update multiqc config with new structural annotation
method
---
assets/sr_multiqc_config.yaml | 107 ++++++++++++++++++++++++++++++++++
1 file changed, 107 insertions(+)
create mode 100644 assets/sr_multiqc_config.yaml
diff --git a/assets/sr_multiqc_config.yaml b/assets/sr_multiqc_config.yaml
new file mode 100644
index 0000000..18076ef
--- /dev/null
+++ b/assets/sr_multiqc_config.yaml
@@ -0,0 +1,107 @@
+report_comment: >
+ This report has been generated by the <a href="https://forgemia.inra.fr/genotoul-bioinfo/metagwgs" target="_blank">genotoul-bioinfo/metagwgs</a>
+ analysis pipeline. For information about how to interpret these results, please see the
+ <a href="https://forgemia.inra.fr/genotoul-bioinfo/metagwgs" target="_blank">documentation</a>.
+
+extra_fn_clean_trim:
+ - "cleaned_"
+ - "raw_"
+ - '_kept_contigs'
+ - '.count_reads_on_contigs'
+ - '.no_filter'
+ - '.host_filter'
+ - '_scaffolds'
+ - '.txt'
+ - '.contigs'
+ - '.sort'
+ - '_kaiju_MEM_verbose'
+ - '_sickle'
+ - '_cutadapt'
+
+extra_fn_clean_exts:
+ - "_select_contigs_cpm"
+
+use_filename_as_sample_name:
+ - cutadapt
+
+module_order:
+ - fastqc:
+ name: 'FastQC (raw)'
+ path_filters_exclude:
+ - '*cleaned_*.zip'
+ - cutadapt
+ - sickle:
+ path_filters:
+ - '*_sickle.log'
+ - samtools:
+ name : 'Reads before host reads filter'
+ info: 'This section reports of the reads alignement against host genome with bwa.'
+ path_filters:
+ - '*.no_filter.flagstat'
+ - samtools:
+ name : 'Reads after host reads filter'
+ info: 'This section reports of the cleaned reads alignement against host genome with bwa.'
+ path_filters:
+ - '*.host_filter.flagstat'
+ - fastqc:
+ name: 'FastQC (cleaned)'
+ info: 'This section of the report shows FastQC results after adapter trimming and cleaning.'
+ path_filters:
+ - '*cleaned_*.zip'
+ - kaiju
+ - quast:
+ name: 'Quast primary assembly'
+ info: 'This section of the report shows quast results after assembly'
+ path_filters:
+ - '*quast_primary/*/report.tsv'
+ - quast:
+ name: 'Quast filtered assembly'
+ info: 'This section of the report shows quast results after filtering of assembly'
+ path_filters:
+ - '*quast_filtered/*/report.tsv'
+ - samtools:
+ name : 'Reads after deduplication'
+ info: 'This section reports of deduplicated reads alignement against contigs with bwa.'
+ path_filters:
+ - '*.count_reads_on_contigs.flagstat'
+ - prokka:
+ name: 'Structural annotation'
+ info: 'This section of the report shows structural annotations results. CDS are predicted using Prodigal, rRNA using Barrnap and tRNA using tRNAscan-se.'
+ - featureCounts
+ - featureCounts
+ - custom_content:
+ name: 'Binning results'
+ info: 'This section of the report shows quast results after binning the contigs into species genomes'
+ path_filters:
+ - '*mqc.tsv'
+
+report_section_order:
+ stats_table:
+ order: -1000
+ bins_quality:
+ order: -1010
+ bins_quality_count:
+ order: -1020
+ bins_quality_size:
+ order: -1030
+ binning_heatmap:
+ order: -1040
+ software_versions:
+ order: -1050
+
+
+
+prokka_fn_snames: True
+prokka_table: True
+
+featurecounts:
+ fn: '*.summary'
+ shared: true
+
+
+table_columns_visible:
+ FastQC (raw):
+ percent_duplicates: False
+ percent_gc: False
+ Structural annotation:
+ organism: False
--
GitLab
From 5875de80a9ded1aa47b790afe034debc67820b64 Mon Sep 17 00:00:00 2001
From: Jean Mainguy <jean.mainguy@inra.fr>
Date: Thu, 20 Oct 2022 23:38:45 +0200
Subject: [PATCH 116/194] simple script to rename contigs with sample and
contig number
---
modules/rename_contigs.nf | 0
1 file changed, 0 insertions(+), 0 deletions(-)
create mode 100644 modules/rename_contigs.nf
diff --git a/modules/rename_contigs.nf b/modules/rename_contigs.nf
new file mode 100644
index 0000000..e69de29
--
GitLab
From bf698c082238cd9a1181768bab31d33158e12020 Mon Sep 17 00:00:00 2001
From: Jean Mainguy <jean.mainguy@inra.fr>
Date: Thu, 20 Oct 2022 23:50:28 +0200
Subject: [PATCH 117/194] call RENAME_CONTIGS in 02_assembly
---
modules/rename_contigs.nf | 0
1 file changed, 0 insertions(+), 0 deletions(-)
delete mode 100644 modules/rename_contigs.nf
diff --git a/modules/rename_contigs.nf b/modules/rename_contigs.nf
deleted file mode 100644
index e69de29..0000000
--
GitLab
From 7356a8f7f9f34b21079f67712091fb88eef0fc40 Mon Sep 17 00:00:00 2001
From: Jean Mainguy <jean.mainguy@inra.fr>
Date: Mon, 24 Oct 2022 14:57:00 +0200
Subject: [PATCH 118/194] add new tools in scrape_software_versions used
instead of prokka
---
bin/scrape_software_versions.py | 9 +++++++--
1 file changed, 7 insertions(+), 2 deletions(-)
diff --git a/bin/scrape_software_versions.py b/bin/scrape_software_versions.py
index e04c4d3..4279474 100755
--- a/bin/scrape_software_versions.py
+++ b/bin/scrape_software_versions.py
@@ -23,7 +23,6 @@ regexes = {
'Hifiasm': ['v_hifiasm_meta.txt', r"ha base version: (\S+)"],
'MetaFlye': ['v_metaflye.txt', r"v(\S+)"],
'Quast': ['v_quast.txt', r"QUAST v(\S+)"],
- 'Prokka': ['v_prokka.txt', r"prokka (\S+)"],
'Kaiju': ['v_kaiju.txt', r"Kaiju (\S+)"],
'Samtools': ['v_samtools.txt', r"samtools (\S+)"],
'Eggnog-Mapper': ['v_eggnogmapper.txt', r"emapper-(\S+)"],
@@ -33,7 +32,10 @@ regexes = {
'CheckM2': ['v_checkm2.txt', r"(\S+)"],
'Metawrap': ['v_metawrap.txt', r"metawrap (\S+)"],
'GTDBTK': ['v_gtdbtk.txt', r"...::: GTDB-Tk v(\S+)"],
- 'dRep': ['v_dRep.txt', r"version (\S+)"]
+ 'dRep': ['v_dRep.txt', r"version (\S+)"],
+ 'tRNAscan-SE': ['v_tRNAscan.txt', r"tRNAscan-SE (\S+)"],
+ 'Barrnap': ['v_barrnap.txt', r"barrnap (\S+)"],
+ 'Prodigal': ['v_prodigal.txt', r"Prodigal (\S+):"],
}
results = OrderedDict()
results['metagWGS'] = '<span style="color:#999999;\">N/A</span>'
@@ -65,6 +67,9 @@ results['Metawrap'] = '<span style="color:#999999;\">N/A</span>'
results['dRep'] = '<span style="color:#999999;\">N/A</span>'
results['GTDBTK'] = '<span style="color:#999999;\">N/A</span>'
results['MultiQC'] = '<span style="color:#999999;\">N/A</span>'
+results['tRNAscan-SE'] = '<span style="color:#999999;\">N/A</span>'
+results['Barrnap'] = '<span style="color:#999999;\">N/A</span>'
+results['Prodigal'] = '<span style="color:#999999;\">N/A</span>'
# Search each file using its regex
for k, v in regexes.items():
--
GitLab
From 2bbf8450f0bfcc25e597c12309c6bcf59c827325 Mon Sep 17 00:00:00 2001
From: Jean Mainguy <jean.mainguy@inra.fr>
Date: Mon, 24 Oct 2022 15:04:34 +0200
Subject: [PATCH 119/194] clean nf scripts
---
main.nf | 5 -----
subworkflows/02_assembly.nf | 8 ++++----
2 files changed, 4 insertions(+), 9 deletions(-)
diff --git a/main.nf b/main.nf
index 75c9f7b..87a6bb4 100644
--- a/main.nf
+++ b/main.nf
@@ -448,14 +448,9 @@ workflow {
ch_prokka_ffn = S04_STRUCTURAL_ANNOT.out.ffn
ch_prokka_faa = S04_STRUCTURAL_ANNOT.out.faa
ch_prokka_gff = S04_STRUCTURAL_ANNOT.out.gff
- // ch_prokka_fna = S04_STRUCTURAL_ANNOT.out.fna
ch_prokka_report = S04_STRUCTURAL_ANNOT.out.report
- // ch_contigs_and_reads = ch_prokka_fna
- // .join(ch_reads, remainder: true)
-
ch_software_versions_S04 = S04_STRUCTURAL_ANNOT.out.software_versions
- // exit 0, 'Exit now... S04_STRUCTURAL_ANNOT being constructed '
}
ch_bam = Channel.empty()
diff --git a/subworkflows/02_assembly.nf b/subworkflows/02_assembly.nf
index 6f6141e..819c674 100644
--- a/subworkflows/02_assembly.nf
+++ b/subworkflows/02_assembly.nf
@@ -6,10 +6,10 @@ include { SAMTOOLS } from '../modules/read_alignment_metrics'
workflow STEP_02_ASSEMBLY {
take:
- reads
- assembly
- has_assembly
- assembly_tool
+ reads
+ assembly
+ has_assembly
+ assembly_tool
main:
ch_assembler_v = Channel.empty()
--
GitLab
From fed02af24a23a0f05fe2ec7e72de490fa3037794 Mon Sep 17 00:00:00 2001
From: Jean Mainguy <jean.mainguy@inra.fr>
Date: Mon, 24 Oct 2022 15:38:20 +0200
Subject: [PATCH 120/194] remove read aln in step 05
---
main.nf | 56 +++++++++++++++++++-----------------
subworkflows/05_alignment.nf | 49 +++++++++++++++----------------
2 files changed, 53 insertions(+), 52 deletions(-)
diff --git a/main.nf b/main.nf
index 87a6bb4..5604044 100644
--- a/main.nf
+++ b/main.nf
@@ -287,7 +287,7 @@ workflow {
ch_kaiju_db = Channel.empty()
ch_eggnog_db = Channel.empty()
ch_taxonomy = Channel.empty()
- ch_diamond = Channel.empty()
+ ch_diamon_db = Channel.empty()
ch_gtbdtk_db = Channel.empty()
@@ -297,7 +297,7 @@ workflow {
ch_kaiju_db = DATABASES.out.kaiju_db
ch_eggnog_db = DATABASES.out.eggnog
ch_taxonomy = DATABASES.out.taxonomy
- ch_diamond = DATABASES.out.diamond
+ ch_diamon_db = DATABASES.out.diamond
ch_gtbdtk_db = DATABASES.out.gtdbtk
ch_multiqc_config = Channel.empty()
@@ -317,7 +317,7 @@ workflow {
ch_final_assembly_flagstat_report = Channel.empty()
ch_assembly_report = Channel.empty()
ch_filtered_report = Channel.empty()
- ch_prokka_report = Channel.empty()
+ ch_annotation_report = Channel.empty()
ch_bins_abundances_report = Channel.empty()
ch_bins_stats_report = Channel.empty()
ch_quast = Channel.empty()
@@ -409,8 +409,8 @@ workflow {
ch_reads = S02_ASSEMBLY.out.reads
ch_bam = S02_ASSEMBLY.out.bam
+ ch_sam_coverage = S02_ASSEMBLY.out.coverage
ch_idxstats = S02_ASSEMBLY.out.idxstats
-
ch_unfilter_assembly_flagstat_report = S02_ASSEMBLY.out.flagstat
ch_quast_before_filter_report = S02_ASSEMBLY.out.assembly_report
@@ -433,47 +433,51 @@ workflow {
ch_min_contigs_cpm
)
ch_assembly = S03_FILTERING.out.selected
+ ch_bam = S03_FILTERING.out.bam
+ ch_sam_coverage = S02_ASSEMBLY.out.coverage
ch_quast_after_filter_report = S03_FILTERING.out.report
-
+ ch_final_assembly_flagstat_report = S03_FILTERING.out.sam_flagstat
+
+
+
+
}
- ch_contigs_and_reads = Channel.empty()
- ch_prokka_ffn = Channel.empty()
- ch_prokka_faa = Channel.empty()
- ch_prokka_gff = Channel.empty()
- ch_prokka_fna = Channel.empty()
+ ch_annotation_ffn = Channel.empty()
+ ch_annotation_faa = Channel.empty()
+ ch_annotation_gff = Channel.empty()
if ( !params.stop_at_clean && !params.stop_at_assembly && !params.stop_at_filtering ) {
S04_STRUCTURAL_ANNOT ( ch_assembly )
- ch_prokka_ffn = S04_STRUCTURAL_ANNOT.out.ffn
- ch_prokka_faa = S04_STRUCTURAL_ANNOT.out.faa
- ch_prokka_gff = S04_STRUCTURAL_ANNOT.out.gff
- ch_prokka_report = S04_STRUCTURAL_ANNOT.out.report
+ ch_annotation_ffn = S04_STRUCTURAL_ANNOT.out.ffn
+ ch_annotation_faa = S04_STRUCTURAL_ANNOT.out.faa
+ ch_annotation_gff = S04_STRUCTURAL_ANNOT.out.gff
+ ch_annotation_report = S04_STRUCTURAL_ANNOT.out.report
ch_software_versions_S04 = S04_STRUCTURAL_ANNOT.out.software_versions
}
- ch_bam = Channel.empty()
+ // ch_bam = Channel.empty()
ch_m8 = Channel.empty()
- ch_sam_coverage = Channel.empty()
+ // ch_sam_coverage = Channel.empty()
if ( !params.stop_at_clean && !params.stop_at_assembly && !params.stop_at_filtering && !params.stop_at_structural_annot ) {
- S05_ALIGNMENT ( ch_contigs_and_reads, ch_prokka_faa, ch_diamond)
- ch_bam = S05_ALIGNMENT.out.bam
+ S05_ALIGNMENT (ch_annotation_faa, ch_diamon_db)
+ // ch_bam = S05_ALIGNMENT.out.bam
ch_m8 = S05_ALIGNMENT.out.m8
- ch_sam_coverage = S05_ALIGNMENT.out.sam_coverage
+ // ch_sam_coverage = S05_ALIGNMENT.out.sam_coverage
- if (!params.skip_filtering){
- // when filtering is skip reads vs assembly remain unchanged so no need to send it to multiqc
- ch_final_assembly_flagstat_report = S05_ALIGNMENT.out.sam_flagstat
- }
+ // if (!params.skip_filtering){
+ // // when filtering is skip reads vs assembly remain unchanged so no need to send it to multiqc
+ // // ch_final_assembly_flagstat_report = S05_ALIGNMENT.out.sam_flagstat
+ // }
ch_software_versions_S05 = S05_ALIGNMENT.out.software_versions
}
ch_quant_report = Channel.empty()
ch_v_eggnogmapper = Channel.empty()
if ( !params.stop_at_clean && !params.stop_at_assembly && !params.stop_at_filtering && !params.stop_at_structural_annot && !params.skip_func_annot ) {
- S06_FUNC_ANNOT ( ch_prokka_ffn, ch_prokka_faa, ch_prokka_gff, ch_bam, ch_m8, ch_eggnog_db )
+ S06_FUNC_ANNOT ( ch_annotation_ffn, ch_annotation_faa, ch_annotation_gff, ch_bam, ch_m8, ch_eggnog_db )
ch_quant_report = S06_FUNC_ANNOT.out.quant_report
ch_software_versions_S06 = S06_FUNC_ANNOT.out.software_versions
@@ -486,7 +490,7 @@ workflow {
}
if ( !params.stop_at_clean && !params.stop_at_assembly && !params.stop_at_filtering && !params.stop_at_structural_annot && !params.skip_binning ) {
- S08_BINNING( ch_reads, ch_prokka_fna, ch_bam, ch_gtbdtk_db, ch_quast )
+ S08_BINNING( ch_reads, ch_annotation_fna, ch_bam, ch_gtbdtk_db, ch_quast )
ch_bins_abundances_report = S08_BINNING.out.bins_abundances_report
ch_bins_stats_report = S08_BINNING.out.bins_stats_report
@@ -515,7 +519,7 @@ workflow {
ch_quast_before_filter_report.collect().ifEmpty([]),
ch_quast_after_filter_report.collect().ifEmpty([]),
ch_final_assembly_flagstat_report.collect().ifEmpty([]),
- ch_prokka_report.collect().ifEmpty([]),
+ ch_annotation_report.collect().ifEmpty([]),
ch_quant_report.collect().ifEmpty([]),
ch_bins_abundances_report.collect().ifEmpty([]),
ch_bins_stats_report.collect().ifEmpty([])
diff --git a/subworkflows/05_alignment.nf b/subworkflows/05_alignment.nf
index 1d7c543..191d20f 100644
--- a/subworkflows/05_alignment.nf
+++ b/subworkflows/05_alignment.nf
@@ -4,34 +4,34 @@ include { DIAMOND } from '../modules/diamond'
workflow STEP_05_ALIGNMENT {
take:
- contigs_and_reads
+ // contigs_and_reads
prokka_faa
diamond
main:
- ch_bwa_mem_v = Channel.empty()
- ch_minimap2_v = Channel.empty()
- ch_samtools_v = Channel.empty()
+ // ch_bwa_mem_v = Channel.empty()
+ // ch_minimap2_v = Channel.empty()
+ // ch_samtools_v = Channel.empty()
ch_diamond_v = Channel.empty()
- publishDir = "05_alignment/05_1_reads_alignment_on_contigs"
- if (params.type == 'SR') {
- BWA_MEM(contigs_and_reads, publishDir)
- ch_bam = BWA_MEM.out.bam
+ // publishDir = "05_alignment/05_1_reads_alignment_on_contigs"
+ // if (params.type == 'SR') {
+ // BWA_MEM(contigs_and_reads, publishDir)
+ // ch_bam = BWA_MEM.out.bam
- ch_bwa_mem_v = BWA_MEM.out.v_bwa_mem2
- ch_samtools_v = BWA_MEM.out.v_samtools
- } else {
- MINIMAP2(contigs_and_reads, publishDir)
- ch_bam = MINIMAP2.out.bam
+ // ch_bwa_mem_v = BWA_MEM.out.v_bwa_mem2
+ // ch_samtools_v = BWA_MEM.out.v_samtools
+ // } else {
+ // MINIMAP2(contigs_and_reads, publishDir)
+ // ch_bam = MINIMAP2.out.bam
- ch_minimap2_v = MINIMAP2.out.v_minimap2
- ch_samtools_v = MINIMAP2.out.v_samtools
- }
+ // ch_minimap2_v = MINIMAP2.out.v_minimap2
+ // ch_samtools_v = MINIMAP2.out.v_samtools
+ // }
- SAMTOOLS(ch_bam, publishDir)
- ch_sam_coverage = SAMTOOLS.out.sam_coverage
- ch_sam_flagstat = SAMTOOLS.out.sam_flagstat
+ // SAMTOOLS(ch_bam, publishDir)
+ // ch_sam_coverage = SAMTOOLS.out.sam_coverage
+ // ch_sam_flagstat = SAMTOOLS.out.sam_flagstat
ch_m8 =Channel.empty()
if (!params.skip_func_annot || !params.skip_taxo_affi){
@@ -42,13 +42,10 @@ workflow STEP_05_ALIGNMENT {
ch_m8 = DIAMOND.out.m8
ch_diamond_v = DIAMOND.out.v_diamond
}
- ch_software_versions = ch_bwa_mem_v.first().mix(ch_minimap2_v.first(),
- ch_samtools_v.first(),
- ch_diamond_v.first())
emit:
- bam = ch_bam
+ // bam = ch_bam
m8 = ch_m8
- sam_coverage = ch_sam_coverage
- software_versions = ch_software_versions
- sam_flagstat = ch_sam_flagstat
+ // sam_coverage = ch_sam_coverage
+ software_versions = ch_diamond_v.first()
+ // sam_flagstat = ch_sam_flagstat
}
--
GitLab
From 9da2e4500118060d6fc822cf98ff464366138f59 Mon Sep 17 00:00:00 2001
From: Jean Mainguy <jean.mainguy@inra.fr>
Date: Mon, 24 Oct 2022 15:39:33 +0200
Subject: [PATCH 121/194] reorganise 02_assembly.nf
---
modules/read_alignment_metrics.nf | 4 +++-
subworkflows/02_assembly.nf | 20 +++++++++-----------
2 files changed, 12 insertions(+), 12 deletions(-)
diff --git a/modules/read_alignment_metrics.nf b/modules/read_alignment_metrics.nf
index e23dc78..ee1bd54 100644
--- a/modules/read_alignment_metrics.nf
+++ b/modules/read_alignment_metrics.nf
@@ -11,12 +11,14 @@ process SAMTOOLS {
tuple val(meta), path("${meta.id}.idxstats"), emit: sam_idxstat
path "${meta.id}.flagstat", emit: sam_flagstat
path "${meta.id}*"
-
+ path "v_samtools.txt", emit: v_samtools
script:
"""
samtools flagstat -@ ${task.cpus} ${bam} > ${meta.id}.flagstat
samtools coverage ${bam} > ${meta.id}.coverage.tsv
samtools idxstats ${bam} > ${meta.id}.idxstats
+
+ samtools --version &> v_samtools.txt
"""
}
\ No newline at end of file
diff --git a/subworkflows/02_assembly.nf b/subworkflows/02_assembly.nf
index 819c674..ee5a207 100644
--- a/subworkflows/02_assembly.nf
+++ b/subworkflows/02_assembly.nf
@@ -64,12 +64,7 @@ workflow STEP_02_ASSEMBLY {
ch_reads = READS_DEDUPLICATION.out.dedup_reads
ch_bam = READS_DEDUPLICATION.out.bam
- SAMTOOLS(ch_bam, "02_assembly")
- ch_idxstats = SAMTOOLS.out.sam_idxstat
- ch_flagstat = SAMTOOLS.out.sam_flagstat
-
ch_bwa_mem_v = READS_DEDUPLICATION.out.v_bwa_mem2
- ch_samtools_v = READS_DEDUPLICATION.out.v_samtools
} else {
@@ -77,16 +72,18 @@ workflow STEP_02_ASSEMBLY {
MINIMAP2(ch_contigs_and_reads,"02_assembly")
ch_bam = MINIMAP2.out.bam
- SAMTOOLS(ch_bam, "02_assembly")
- ch_idxstats = SAMTOOLS.out.sam_idxstat
- ch_flagstat = SAMTOOLS.out.sam_flagstat
-
ch_minimap2_v = MINIMAP2.out.v_minimap2
- ch_samtools_v = MINIMAP2.out.v_samtools
+
}
-
}
+ SAMTOOLS(ch_bam, "02_assembly")
+
+ ch_idxstats = SAMTOOLS.out.sam_idxstat
+ ch_flagstat = SAMTOOLS.out.sam_flagstat
+ ch_coverage = SAMTOOLS.out.sam_coverage
+ ch_samtools_v = SAMTOOLS.out.v_samtools
+
ch_software_versions = ch_assembler_v.first().mix( ch_quast_v.first(),
ch_bwa_mem_v.first(),
ch_minimap2_v.first(),
@@ -99,6 +96,7 @@ workflow STEP_02_ASSEMBLY {
bam = ch_bam
idxstats = ch_idxstats
flagstat = ch_flagstat
+ coverage = ch_coverage
assembly_report = ch_assembly_report
software_versions = ch_software_versions
}
\ No newline at end of file
--
GitLab
From e25e010be22c682fdd05e0e95c814b00aafd3f36 Mon Sep 17 00:00:00 2001
From: Jean Mainguy <jean.mainguy@inra.fr>
Date: Mon, 24 Oct 2022 15:59:42 +0200
Subject: [PATCH 122/194] rename diamond result channel for better clarity
---
main.nf | 10 +++++-----
1 file changed, 5 insertions(+), 5 deletions(-)
diff --git a/main.nf b/main.nf
index 5604044..173bf84 100644
--- a/main.nf
+++ b/main.nf
@@ -434,7 +434,7 @@ workflow {
)
ch_assembly = S03_FILTERING.out.selected
ch_bam = S03_FILTERING.out.bam
- ch_sam_coverage = S02_ASSEMBLY.out.coverage
+ ch_sam_coverage = S03_FILTERING.out.coverage
ch_quast_after_filter_report = S03_FILTERING.out.report
ch_final_assembly_flagstat_report = S03_FILTERING.out.sam_flagstat
@@ -458,13 +458,13 @@ workflow {
}
// ch_bam = Channel.empty()
- ch_m8 = Channel.empty()
+ ch_diamond_result = Channel.empty()
// ch_sam_coverage = Channel.empty()
if ( !params.stop_at_clean && !params.stop_at_assembly && !params.stop_at_filtering && !params.stop_at_structural_annot ) {
S05_ALIGNMENT (ch_annotation_faa, ch_diamon_db)
// ch_bam = S05_ALIGNMENT.out.bam
- ch_m8 = S05_ALIGNMENT.out.m8
+ ch_diamond_result = S05_ALIGNMENT.out.m8
// ch_sam_coverage = S05_ALIGNMENT.out.sam_coverage
// if (!params.skip_filtering){
@@ -477,14 +477,14 @@ workflow {
ch_quant_report = Channel.empty()
ch_v_eggnogmapper = Channel.empty()
if ( !params.stop_at_clean && !params.stop_at_assembly && !params.stop_at_filtering && !params.stop_at_structural_annot && !params.skip_func_annot ) {
- S06_FUNC_ANNOT ( ch_annotation_ffn, ch_annotation_faa, ch_annotation_gff, ch_bam, ch_m8, ch_eggnog_db )
+ S06_FUNC_ANNOT ( ch_annotation_ffn, ch_annotation_faa, ch_annotation_gff, ch_bam, ch_diamond_result, ch_eggnog_db )
ch_quant_report = S06_FUNC_ANNOT.out.quant_report
ch_software_versions_S06 = S06_FUNC_ANNOT.out.software_versions
}
if ( !params.stop_at_clean && !params.stop_at_assembly && !params.stop_at_filtering && !params.stop_at_structural_annot && !params.skip_taxo_affi ) {
- S07_TAXO_AFFI ( ch_taxonomy, ch_m8, ch_sam_coverage)
+ S07_TAXO_AFFI ( ch_taxonomy, ch_diamond_result, ch_sam_coverage)
ch_software_versions_S07 = S07_TAXO_AFFI.out.software_versions
}
--
GitLab
From b9a51f32ce0e39928a6a5b3d8b6984cc25721602 Mon Sep 17 00:00:00 2001
From: Jean Mainguy <jean.mainguy@inra.fr>
Date: Mon, 24 Oct 2022 16:00:02 +0200
Subject: [PATCH 123/194] rm graph as expected output
---
modules/assign_taxonomy.nf | 1 -
1 file changed, 1 deletion(-)
diff --git a/modules/assign_taxonomy.nf b/modules/assign_taxonomy.nf
index 9d765ad..7fd509d 100644
--- a/modules/assign_taxonomy.nf
+++ b/modules/assign_taxonomy.nf
@@ -11,7 +11,6 @@ process ASSIGN_TAXONOMY {
tuple val(meta.id), path("${meta.id}.percontig.tsv"), emit: t_percontig
tuple val(meta.id), path("${meta.id}.pergene.tsv"), emit: t_pergene
tuple val(meta.id), path("${meta.id}.warn.tsv"), emit: t_warn
- tuple val(meta.id), path("graphs"), emit: t_graphs
path "${meta.id}_quantif_percontig.tsv", emit: q_all
path "${meta.id}_quantif_percontig_by_superkingdom.tsv", emit: q_superkingdom
path "${meta.id}_quantif_percontig_by_phylum.tsv", emit: q_phylum
--
GitLab
From d1be56c6a1c705a06ade856b91dd59e593e93928 Mon Sep 17 00:00:00 2001
From: Jean Mainguy <jean.mainguy@inra.fr>
Date: Mon, 24 Oct 2022 16:53:27 +0200
Subject: [PATCH 124/194] refactoring of filter_contig_per_cpm.py using pyfastx
#203
---
bin/Filter_contig_per_cpm.py | 95 --------------------------------
bin/filter_contig_per_cpm.py | 103 +++++++++++++++++++++++++++++++++++
conf/base.config | 4 +-
conf/test.config | 2 +-
main.nf | 23 +++-----
modules/filtering_cpm.nf | 2 +-
subworkflows/03_filtering.nf | 20 +++++--
7 files changed, 131 insertions(+), 118 deletions(-)
delete mode 100755 bin/Filter_contig_per_cpm.py
create mode 100755 bin/filter_contig_per_cpm.py
diff --git a/bin/Filter_contig_per_cpm.py b/bin/Filter_contig_per_cpm.py
deleted file mode 100755
index 9127e57..0000000
--- a/bin/Filter_contig_per_cpm.py
+++ /dev/null
@@ -1,95 +0,0 @@
-#!/usr/bin/env python
-
-"""----------------------------------------------------------------------------
- Script Name: Filter_contig_per_cpm.py
- Description: Calculates the CPM normalization of mapped reads for each \
- contig and returns contigs which have a CPM > cutoff in .fa.
- Input files: Samtools idxstats output file, .fasta file of contigs.
- Created By: Joanna Fourquet
- Date: 2020-04-01
--------------------------------------------------------------------------------
-"""
-
-# Metadata
-__author__ = 'Joanna Fourquet \
-- GenPhySE - Team NED'
-__copyright__ = 'Copyright (C) 2020 INRA'
-__license__ = 'GNU General Public License'
-__version__ = '0.1'
-__email__ = 'support.bioinfo.genotoul@inra.fr'
-__status__ = 'dev'
-
-# Status: dev
-
-# Modules importation
-
-try:
- import argparse
- import sys
- import pandas as p
- import numpy as np
- from Bio.Seq import Seq
- from Bio.SeqRecord import SeqRecord
- import pprint
- from Bio import SeqIO
-except ImportError as error:
- print(error)
- exit(1)
-
-################################################
-# Function
-################################################
-
-def cpm(counts):
- N = np.sum(counts.iloc[:,2], axis=0)
- C = counts.iloc[:,2]
- cpm_values = 1e6 * C / N
- return(cpm_values)
-
-def main(argv):
- parser = argparse.ArgumentParser()
- parser.add_argument("-i", "--samtools_idxstats", \
- required = True, help = "samtools idxstats file containing contig id, \
- sequence length, number of mapped reads or fragments, \
- number of unmapped reads or fragments")
- parser.add_argument('-f', '--fasta_file', required = True, help = \
- 'fasta file containing sequences of contigs.')
- parser.add_argument("-c", "--cutoff_cpm", required = True, \
- help = "Minimum number of reads in a contig")
- parser.add_argument("-s", "--select", \
- help = "Name of outpout .fa file containing contigs which passed cpm cutoff")
- parser.add_argument("-d", "--discard", \
- help = "Name of outpout .fa file containing contigs which don't passed cpm cutoff")
- args = parser.parse_args()
-
- # Read input table
- raw_counts = p.read_table(args.samtools_idxstats,sep ='\t',header = None, comment="*")
-
- # Calculates cpm for each contig
- res_cpm = cpm(raw_counts)
-
- cutoff = float(args.cutoff_cpm)
- # Contigs with nb reads > cutoff
- kept_contigs = raw_counts.iloc[np.where(res_cpm >= cutoff)[0],0]
-
- # Contigs with nb reads < cutoff
- unkept_contigs = raw_counts.iloc[np.where(res_cpm < cutoff)[0],0]
-
- # Write new fasta files with kept and unkept contigs
- with open(args.fasta_file, "rU") as fasta_file,\
- open(args.select, "w") as out_select_handle,\
- open(args.discard, "w") as out_discard_handle:
- for record in SeqIO.parse(fasta_file, "fasta"):
- try :
- contig_id = record.id
- if(contig_id in list(kept_contigs)):
- SeqIO.write(record, out_select_handle, "fasta")
- else:
- if(contig_id in list(unkept_contigs)):
- SeqIO.write(record, out_discard_handle, "fasta")
- except :
- print ("Warning input fasta file: contig " + record.id + " issue")
- pass
-
-if __name__ == "__main__":
- main(sys.argv[1:])
diff --git a/bin/filter_contig_per_cpm.py b/bin/filter_contig_per_cpm.py
new file mode 100755
index 0000000..ba563ec
--- /dev/null
+++ b/bin/filter_contig_per_cpm.py
@@ -0,0 +1,103 @@
+#!/usr/bin/env python
+
+"""----------------------------------------------------------------------------
+ Script Name: Filter_contig_per_cpm.py
+ Description: Calculates the CPM normalization of mapped reads for each \
+ contig and returns contigs which have a CPM > cutoff in .fa.
+ Input files: Samtools idxstats output file, .fasta file of contigs.
+ Created By: Joanna Fourquet
+ Date: 2020-04-01
+-------------------------------------------------------------------------------
+"""
+
+# Metadata
+__author__ = 'Joanna Fourquet \
+- GenPhySE - Team NED'
+__copyright__ = 'Copyright (C) 2020 INRA'
+__license__ = 'GNU General Public License'
+__version__ = '0.1'
+__email__ = 'support.bioinfo.genotoul@inra.fr'
+__status__ = 'dev'
+
+# Status: dev
+
+# Modules importation
+
+
+from argparse import ArgumentParser, ArgumentDefaultsHelpFormatter
+import sys
+import pandas as pd
+import numpy as np
+import logging
+from Bio import SeqIO
+import pyfastx
+
+################################################
+# Function
+################################################
+
+def parse_arguments():
+ """Parse script arguments."""
+ parser = ArgumentParser(description="...",
+ formatter_class=ArgumentDefaultsHelpFormatter)
+
+ parser.add_argument("-i", "--samtools_idxstats", required = True,
+ help = "samtools idxstats file containing contig id, \
+ sequence length, number of mapped reads or fragments, \
+ number of unmapped reads or fragments")
+
+ parser.add_argument('-f', '--fasta_file', required = True,
+ help = 'fasta file containing sequences of contigs.')
+ parser.add_argument("-c", "--cutoff_cpm", required = True,
+ help = "Minimum number of reads in a contig")
+ parser.add_argument("-s", "--select",
+ help = "Name of outpout .fa file containing contigs which passed cpm cutoff")
+ parser.add_argument("-d", "--discard",
+ help = "Name of outpout .fa file containing contigs which don't passed cpm cutoff")
+
+
+ parser.add_argument("-v", "--verbose", help="increase output verbosity",
+ action="store_true")
+
+ args = parser.parse_args()
+ return args
+
+def cpm(counts):
+ N = np.sum(counts.iloc[:,2], axis=0)
+ C = counts.iloc[:,2]
+ cpm_values = 1e6 * C / N
+ return(cpm_values)
+
+def main(argv):
+ args = parse_arguments()
+
+ if args.verbose:
+ logging.basicConfig(format="%(levelname)s: %(message)s", level=logging.DEBUG)
+ logging.info('Mode verbose ON')
+
+ else:
+ logging.basicConfig(format="%(levelname)s: %(message)s")
+
+
+ # Read input table
+ raw_counts = pd.read_table(args.samtools_idxstats,sep ='\t',header = None, comment="*")
+
+ # Calculates cpm for each contig
+ res_cpm = cpm(raw_counts)
+
+ cutoff = float(args.cutoff_cpm)
+ # Contigs with nb reads > cutoff
+ kept_contigs = raw_counts.iloc[np.where(res_cpm >= cutoff)[0],0]
+
+
+ # Write new fasta files with kept and unkept contigs
+ with open(args.select, "w") as out_select_handle, open(args.discard, "w") as out_discard_handle:
+
+ for contig, seq in pyfastx.Fasta(args.fasta_file, build_index=False):
+ if contig in kept_contigs:
+ out_select_handle.write(f'>{contig}\n{seq}\n')
+ else:
+ out_discard_handle.write(f'>{contig}\n{seq}\n')
+
+if __name__ == "__main__":
+ main(sys.argv[1:])
diff --git a/conf/base.config b/conf/base.config
index dd61115..721713b 100644
--- a/conf/base.config
+++ b/conf/base.config
@@ -55,8 +55,8 @@ process {
memory = { 32.GB * task.attempt }
}
withLabel: ASSEMBLY_FILTER {
- memory = { 8.GB * task.attempt }
- cpus = 4
+ memory = { 2.GB * task.attempt }
+ cpus = 1
}
withLabel: CD_HIT {
memory = { 50.GB * task.attempt }
diff --git a/conf/test.config b/conf/test.config
index ae50b57..8b437cd 100644
--- a/conf/test.config
+++ b/conf/test.config
@@ -47,7 +47,7 @@ process {
}
withLabel: ASSEMBLY_FILTER {
memory = { 1.GB * task.attempt }
- cpus = 2
+ cpus = 1
}
withLabel: CD_HIT {
memory = { 16.GB * task.attempt }
diff --git a/main.nf b/main.nf
index 173bf84..2c53770 100644
--- a/main.nf
+++ b/main.nf
@@ -418,28 +418,23 @@ workflow {
}
if ( !params.stop_at_clean && !params.stop_at_assembly && !params.skip_filtering ) {
+ print("IN FILTERING")
ch_min_contigs_cpm = Channel.value(params.min_contigs_cpm)
- ch_assembly
- .splitFasta(by: 100000, file: true)
- .set{ch_chunk_assembly_for_filter}
- ch_chunk_assembly_for_filter
- .combine(ch_idxstats, by:0)
- .set{ch_assembly_and_idxstats}
S03_FILTERING (
- ch_assembly_and_idxstats,
+ ch_assembly,
+ ch_idxstats,
+ ch_bam,
ch_min_contigs_cpm
)
- ch_assembly = S03_FILTERING.out.selected
+
+ ch_assembly = S03_FILTERING.out.selected_contigs
ch_bam = S03_FILTERING.out.bam
- ch_sam_coverage = S03_FILTERING.out.coverage
- ch_quast_after_filter_report = S03_FILTERING.out.report
- ch_final_assembly_flagstat_report = S03_FILTERING.out.sam_flagstat
-
-
-
+ // ch_sam_coverage = S03_FILTERING.out.coverage
+ ch_quast_after_filter_report = S03_FILTERING.out.quast_report
+ // ch_final_assembly_flagstat_report = S03_FILTERING.out.sam_flagstat
}
diff --git a/modules/filtering_cpm.nf b/modules/filtering_cpm.nf
index 951a201..0155034 100644
--- a/modules/filtering_cpm.nf
+++ b/modules/filtering_cpm.nf
@@ -12,7 +12,7 @@ process CHUNK_ASSEMBLY_FILTER {
script:
chunk_name = assembly_file.baseName
"""
- Filter_contig_per_cpm.py -i ${idxstats} -f ${assembly_file} -c ${min_cpm} -s ${chunk_name}_select_cpm${min_cpm}.fasta -d ${chunk_name}_discard_cpm${min_cpm}.fasta
+ filter_contig_per_cpm.py -i ${idxstats} -f ${assembly_file} -c ${min_cpm} -s ${chunk_name}_select_cpm${min_cpm}.fasta -d ${chunk_name}_discard_cpm${min_cpm}.fasta
"""
}
diff --git a/subworkflows/03_filtering.nf b/subworkflows/03_filtering.nf
index b13e490..488a576 100644
--- a/subworkflows/03_filtering.nf
+++ b/subworkflows/03_filtering.nf
@@ -3,12 +3,21 @@ include { QUAST as FILTERED_QUAST } from '../modules/metaquast'
workflow STEP_03_ASSEMBLY_FILTER {
take:
- assembly_and_idxstats
+ assemblies
+ idxstats
+ bam
min_cpm
main:
+ ch_chunk_assembly_for_filter = assemblies
+ .splitFasta(by: 100000, file: true)
+
+
+ ch_assembly_and_idxstats = ch_chunk_assembly_for_filter
+ .combine(idxstats, by:0)
+
CHUNK_ASSEMBLY_FILTER (
- assembly_and_idxstats,
+ ch_assembly_and_idxstats,
min_cpm
)
ch_chunk_selected = CHUNK_ASSEMBLY_FILTER.out.chunk_selected
@@ -30,10 +39,11 @@ workflow STEP_03_ASSEMBLY_FILTER {
ch_merged_selected = MERGE_ASSEMBLY_FILTER.out.merged_selected
FILTERED_QUAST( ch_merged_selected, "03_filtering/quast_filtered" )
- ch_filtered_report = FILTERED_QUAST.out.report
+ ch_quast_report = FILTERED_QUAST.out.report
emit:
- selected = ch_merged_selected
- report = ch_filtered_report
+ selected_contigs = ch_merged_selected
+ quast_report = ch_quast_report
+ bam = bam
}
\ No newline at end of file
--
GitLab
From 6d8e3b2eeb2cf070fd09693379d03910ff5a2f8e Mon Sep 17 00:00:00 2001
From: Jean Mainguy <jean.mainguy@inra.fr>
Date: Mon, 24 Oct 2022 18:20:34 +0200
Subject: [PATCH 125/194] compelete refactoring of filter_contig_per_cpm.py
---
bin/filter_contig_per_cpm.py | 42 +++++++++++++++++++-----------------
1 file changed, 22 insertions(+), 20 deletions(-)
diff --git a/bin/filter_contig_per_cpm.py b/bin/filter_contig_per_cpm.py
index ba563ec..8f01cec 100755
--- a/bin/filter_contig_per_cpm.py
+++ b/bin/filter_contig_per_cpm.py
@@ -2,18 +2,17 @@
"""----------------------------------------------------------------------------
Script Name: Filter_contig_per_cpm.py
- Description: Calculates the CPM normalization of mapped reads for each \
+ Description: Calculates the CPM normalization of mapped reads for each
contig and returns contigs which have a CPM > cutoff in .fa.
Input files: Samtools idxstats output file, .fasta file of contigs.
- Created By: Joanna Fourquet
- Date: 2020-04-01
+ Created By: Jean Mainguy
+ Date: 2022-24-10
-------------------------------------------------------------------------------
"""
# Metadata
-__author__ = 'Joanna Fourquet \
-- GenPhySE - Team NED'
-__copyright__ = 'Copyright (C) 2020 INRA'
+__author__ = 'Mainguy Jean - Plateforme bioinformatique Toulouse'
+__copyright__ = 'Copyright (C) 2022 INRAE'
__license__ = 'GNU General Public License'
__version__ = '0.1'
__email__ = 'support.bioinfo.genotoul@inra.fr'
@@ -25,11 +24,9 @@ __status__ = 'dev'
from argparse import ArgumentParser, ArgumentDefaultsHelpFormatter
-import sys
import pandas as pd
import numpy as np
import logging
-from Bio import SeqIO
import pyfastx
################################################
@@ -62,13 +59,8 @@ def parse_arguments():
args = parser.parse_args()
return args
-def cpm(counts):
- N = np.sum(counts.iloc[:,2], axis=0)
- C = counts.iloc[:,2]
- cpm_values = 1e6 * C / N
- return(cpm_values)
-def main(argv):
+def main():
args = parse_arguments()
if args.verbose:
@@ -78,18 +70,28 @@ def main(argv):
else:
logging.basicConfig(format="%(levelname)s: %(message)s")
+ cpm_cutoff = float(args.cutoff_cpm)
# Read input table
- raw_counts = pd.read_table(args.samtools_idxstats,sep ='\t',header = None, comment="*")
+ idxstat_df = pd.read_csv(args.samtools_idxstats,
+ sep ='\t',
+ names = ['reference_sequence_name',
+ 'sequence_length',
+ '# mapped_read-segments',
+ '#_unmapped_read-segments'],
+ comment="*")
# Calculates cpm for each contig
- res_cpm = cpm(raw_counts)
+ sum_reads = idxstat_df['# mapped_read-segments'].sum()
- cutoff = float(args.cutoff_cpm)
- # Contigs with nb reads > cutoff
- kept_contigs = raw_counts.iloc[np.where(res_cpm >= cutoff)[0],0]
+ logging.info(f'With a cpm cutoff of {args.cutoff_cpm}, contigs with less than {(sum_reads*cpm_cutoff)/1e6} reads are removed.')
+ idxstat_df['cpm_count'] = 1e6 * idxstat_df['# mapped_read-segments']/sum_reads
+
+ # Contigs with nb reads > cutoff
+ kept_contigs = idxstat_df.loc[idxstat_df["cpm_count"] >= cpm_cutoff, "reference_sequence_name"].to_list()
+ logging.info(f'{len(kept_contigs)}/{len(idxstat_df)} contigs are kept with a cpm cutoff of {cpm_cutoff}.')
# Write new fasta files with kept and unkept contigs
with open(args.select, "w") as out_select_handle, open(args.discard, "w") as out_discard_handle:
@@ -100,4 +102,4 @@ def main(argv):
out_discard_handle.write(f'>{contig}\n{seq}\n')
if __name__ == "__main__":
- main(sys.argv[1:])
+ main()
--
GitLab
From 57a6984067a434b3a3babb1bd6d0bb3c60b59e36 Mon Sep 17 00:00:00 2001
From: Jean Mainguy <jean.mainguy@inra.fr>
Date: Mon, 24 Oct 2022 19:00:10 +0200
Subject: [PATCH 126/194] add possibility to process multiple idxstat in
filter_contig_per_cpm.py
---
bin/filter_contig_per_cpm.py | 53 ++++++++++++++++++++++++++----------
1 file changed, 39 insertions(+), 14 deletions(-)
diff --git a/bin/filter_contig_per_cpm.py b/bin/filter_contig_per_cpm.py
index 8f01cec..fdea68d 100755
--- a/bin/filter_contig_per_cpm.py
+++ b/bin/filter_contig_per_cpm.py
@@ -38,16 +38,16 @@ def parse_arguments():
parser = ArgumentParser(description="...",
formatter_class=ArgumentDefaultsHelpFormatter)
- parser.add_argument("-i", "--samtools_idxstats", required = True,
+ parser.add_argument("-i", "--samtools_idxstats", nargs='+', required = True,
help = "samtools idxstats file containing contig id, \
sequence length, number of mapped reads or fragments, \
number of unmapped reads or fragments")
- parser.add_argument('-f', '--fasta_file', required = True,
+ parser.add_argument('-f', '--fasta_file', required = True,
help = 'fasta file containing sequences of contigs.')
parser.add_argument("-c", "--cutoff_cpm", required = True,
help = "Minimum number of reads in a contig")
- parser.add_argument("-s", "--select",
+ parser.add_argument("-s", "--select",
help = "Name of outpout .fa file containing contigs which passed cpm cutoff")
parser.add_argument("-d", "--discard",
help = "Name of outpout .fa file containing contigs which don't passed cpm cutoff")
@@ -59,6 +59,36 @@ def parse_arguments():
args = parser.parse_args()
return args
+def combine_idxstat_files(idxstat_files):
+ """
+ Combine multiple idxstat files that have the same contigs.
+
+ Sum the #_mapped_read_segments column over multiple idxstat files that have the same reference sequences.
+ """
+ columns_names = ['reference_sequence_name',
+ 'sequence_length',
+ '#_mapped_read_segments',
+ '#_unmapped_read-segments']
+
+ idxstat_df = pd.read_csv(idxstat_files[0],
+ sep ='\t',
+ names = columns_names,
+ usecols = ['reference_sequence_name',
+ 'sequence_length',
+ '#_mapped_read_segments',],
+ comment="*").set_index('reference_sequence_name')
+
+ for idxstat_file in idxstat_files[1:]:
+ other_idxstat_df = pd.read_csv(idxstat_file,
+ sep ='\t',
+ names = columns_names,
+ usecols = ['reference_sequence_name',
+ '#_mapped_read_segments',],
+ comment="*").set_index('reference_sequence_name')
+
+ idxstat_df['#_mapped_read_segments'] += other_idxstat_df['#_mapped_read_segments']
+
+ return idxstat_df
def main():
args = parse_arguments()
@@ -72,24 +102,19 @@ def main():
cpm_cutoff = float(args.cutoff_cpm)
- # Read input table
- idxstat_df = pd.read_csv(args.samtools_idxstats,
- sep ='\t',
- names = ['reference_sequence_name',
- 'sequence_length',
- '# mapped_read-segments',
- '#_unmapped_read-segments'],
- comment="*")
+ # Read input tables
+ idxstat_df = combine_idxstat_files(args.samtools_idxstats)
# Calculates cpm for each contig
- sum_reads = idxstat_df['# mapped_read-segments'].sum()
+ sum_reads = idxstat_df['#_mapped_read_segments'].sum()
+ logging.info(f'Total number of mapped reads {sum_reads}')
logging.info(f'With a cpm cutoff of {args.cutoff_cpm}, contigs with less than {(sum_reads*cpm_cutoff)/1e6} reads are removed.')
- idxstat_df['cpm_count'] = 1e6 * idxstat_df['# mapped_read-segments']/sum_reads
+ idxstat_df['cpm_count'] = 1e6 * idxstat_df['#_mapped_read_segments']/sum_reads
# Contigs with nb reads > cutoff
- kept_contigs = idxstat_df.loc[idxstat_df["cpm_count"] >= cpm_cutoff, "reference_sequence_name"].to_list()
+ kept_contigs = idxstat_df.loc[idxstat_df["cpm_count"] >= cpm_cutoff].index
logging.info(f'{len(kept_contigs)}/{len(idxstat_df)} contigs are kept with a cpm cutoff of {cpm_cutoff}.')
# Write new fasta files with kept and unkept contigs
--
GitLab
From a0536948b421b872c9b413e517caac8681bca6f6 Mon Sep 17 00:00:00 2001
From: Jean Mainguy <jean.mainguy@inra.fr>
Date: Tue, 25 Oct 2022 12:01:03 +0200
Subject: [PATCH 127/194] add structure and process to rescue reads in
discarded contigs
---
subworkflows/03_filtering.nf | 125 +++++++++++++++++++++++++++++++++--
1 file changed, 120 insertions(+), 5 deletions(-)
diff --git a/subworkflows/03_filtering.nf b/subworkflows/03_filtering.nf
index 488a576..dab2324 100644
--- a/subworkflows/03_filtering.nf
+++ b/subworkflows/03_filtering.nf
@@ -1,5 +1,7 @@
include { CHUNK_ASSEMBLY_FILTER; MERGE_ASSEMBLY_FILTER} from '../modules/filtering_cpm.nf'
-include { QUAST as FILTERED_QUAST } from '../modules/metaquast'
+include { QUAST } from '../modules/metaquast'
+include { MINIMAP2; BWA_MEM } from '../modules/read_alignment'
+include { SAMTOOLS } from '../modules/read_alignment_metrics'
workflow STEP_03_ASSEMBLY_FILTER {
take:
@@ -37,13 +39,126 @@ workflow STEP_03_ASSEMBLY_FILTER {
min_cpm
)
ch_merged_selected = MERGE_ASSEMBLY_FILTER.out.merged_selected
+ discarded_contigs = MERGE_ASSEMBLY_FILTER.out.merged_discarded
- FILTERED_QUAST( ch_merged_selected, "03_filtering/quast_filtered" )
- ch_quast_report = FILTERED_QUAST.out.report
+ discarded_contigs_category = discarded_contigs.branch{
+ without: it[1].isEmpty()
+ with: !it[1].isEmpty()
+ }
+ // discarded_contigs_category.view()
+ samples_with_discarded_contigs = discarded_contigs_category.with
+ samples_without_discarded_contigs = discarded_contigs_category.without
+
+ discarded_contigs_category.without.view { "WITHOUT::::::::: > $it" }
+ discarded_contigs_category.with.view { "WITH::::::::: > $it" }
+
+ ch_discarded_contigs_and_bam = samples_with_discarded_contigs.join(bam)
+ ch_discarded_contigs_and_bam.view()
+
+ FILTER_BAM(ch_discarded_contigs_and_bam)
+
+ ch_discarded_bam = FILTER_BAM.out.discarded_contigs_bam
+ ch_selected_bam = FILTER_BAM.out.selected_contigs_bam
+
+ if ( params.type.toUpperCase() == "SR" ) {
+
+ ch_discarded_reads = EXTRACT_PAIRED_READS(ch_discarded_bam)
+
+ ch_contigs_and_discarded_reads = ch_merged_selected.join(ch_discarded_reads)
+
+ BWA_MEM(ch_contigs_and_discarded_reads, "03_filtering")
+ ch_bam_with_discarded_reads = BWA_MEM.out.bam
+
+ } else {
+ ch_discarded_reads = EXTRACT_SINGLE_READS(ch_discarded_bam)
+ ch_contigs_and_discarded_reads = ch_merged_selected.join(ch_discarded_reads)
+ MINIMAP2(ch_contigs_and_reads, "03_filtering")
+ ch_bam_with_discarded_reads = MINIMAP2.out.bam
+
+ }
+
+
+ // ch_merged_bam = MERGE_BAM_FILES(ch_filtered_bam, ch_bam_with_discarded_reads)
+ // SAMTOOLS(ch_merged_bam, '03_filtering/')
+
+
+ ch_merged_bam = Channel.empty()
+ ch_coverage = Channel.empty()
+
+ ch_flagstat= Channel.empty()
+
+ // ch_flagstat = SAMTOOLS.out.sam_flagstat
+ // ch_coverage = SAMTOOLS.out.sam_coverage
+
+ QUAST( ch_merged_selected, "03_filtering/quast_filtered" )
+ ch_quast_report = QUAST.out.report
emit:
selected_contigs = ch_merged_selected
quast_report = ch_quast_report
- bam = bam
-}
\ No newline at end of file
+ bam = ch_merged_bam
+ sam_coverage = ch_coverage
+ sam_flagstat = ch_flagstat
+
+}
+
+
+process FILTER_BAM {
+ tag "${meta.id}"
+
+ input:
+ tuple val(meta), path(discarded_contigs), path(bam), path(bai)
+
+ output:
+ tuple val(meta), path("selected_contigs.bam"), emit: selected_contigs_bam
+ tuple val(meta), path("discarded_contigs.bam"), emit: discarded_contigs_bam
+
+
+ script:
+ """
+
+ samtools faidx $discarded_contigs
+ awk 'BEGIN {FS="\\t"}; {print \$1 FS "0" FS \$2}' ${discarded_contigs}.fai > discarded_contigs.bed
+
+
+ samtools view -@ ${task.cpus} -bL discarded_contigs.bed -U selected_contigs.bam $bam -o discarded_contigs.bam
+
+
+ """
+}
+
+process EXTRACT_PAIRED_READS {
+ tag "${meta.id}"
+
+ input:
+ tuple val(meta), path(bam) //, path(bai)
+
+ output:
+ tuple val(meta), path("reads*.fastq.gz"), emit: reads
+
+
+ script:
+ """
+
+ samtools fastq -@ ${task.cpus} -N -1 reads_R1.fastq.gz -2 reads_R2.fastq.gz $bam
+
+ """
+}
+
+process EXTRACT_SINGLE_READS {
+ tag "${meta.id}"
+
+ input:
+ tuple val(meta), path(bam), path(bai)
+
+ output:
+ tuple val(meta), path("reads.fastq.gz"), emit: reads
+
+ script:
+ """
+
+ samtools fastq -@ ${task.cpus} -N -1 reads.fastq.gz $bam
+
+ """
+}
--
GitLab
From 2850a2f933c36900a564e719f531edbad94bd267 Mon Sep 17 00:00:00 2001
From: Jean Mainguy <jean.mainguy@inra.fr>
Date: Tue, 25 Oct 2022 16:28:37 +0200
Subject: [PATCH 128/194] finish rescuing reads mapping to discareded contigs
---
subworkflows/03_filtering.nf | 54 +++++++++++++++++++++++++++---------
1 file changed, 41 insertions(+), 13 deletions(-)
diff --git a/subworkflows/03_filtering.nf b/subworkflows/03_filtering.nf
index dab2324..97399ef 100644
--- a/subworkflows/03_filtering.nf
+++ b/subworkflows/03_filtering.nf
@@ -46,14 +46,16 @@ workflow STEP_03_ASSEMBLY_FILTER {
with: !it[1].isEmpty()
}
- // discarded_contigs_category.view()
- samples_with_discarded_contigs = discarded_contigs_category.with
- samples_without_discarded_contigs = discarded_contigs_category.without
+
+ samples_without_discarded_contigs = discarded_contigs_category.without.map{ it -> it[0]}
+ samples_without_discarded_contigs.view{"samples WITHOUT DISCARDED CONTIGS: $it"}
+ ch_bam_of_sample_without_discarded_contigs = samples_without_discarded_contigs.join(bam)
+ ch_bam_of_sample_without_discarded_contigs.view{"samples WITHOUT DISCARDED CONTIGS AND bam: $it"}
discarded_contigs_category.without.view { "WITHOUT::::::::: > $it" }
discarded_contigs_category.with.view { "WITH::::::::: > $it" }
- ch_discarded_contigs_and_bam = samples_with_discarded_contigs.join(bam)
+ ch_discarded_contigs_and_bam = discarded_contigs_category.with.join(bam)
ch_discarded_contigs_and_bam.view()
FILTER_BAM(ch_discarded_contigs_and_bam)
@@ -79,17 +81,24 @@ workflow STEP_03_ASSEMBLY_FILTER {
}
- // ch_merged_bam = MERGE_BAM_FILES(ch_filtered_bam, ch_bam_with_discarded_reads)
- // SAMTOOLS(ch_merged_bam, '03_filtering/')
+ // remove bai from ch_bam_with_discarded_reads
+ ch_bam_to_merge = ch_bam_with_discarded_reads.map{ it -> [it[0], it[1]]}.join(ch_selected_bam)
+
+
+
+
+
+ ch_merged_bam = MERGE_BAM_FILES(ch_bam_to_merge)
+ ch_final_bam = ch_bam_of_sample_without_discarded_contigs.mix(ch_merged_bam)
- ch_merged_bam = Channel.empty()
- ch_coverage = Channel.empty()
- ch_flagstat= Channel.empty()
+ ch_final_bam.view { "FINAL BAMMMM::::::::: > $it" }
- // ch_flagstat = SAMTOOLS.out.sam_flagstat
- // ch_coverage = SAMTOOLS.out.sam_coverage
+ SAMTOOLS(ch_final_bam, '03_filtering/')
+
+ ch_flagstat = SAMTOOLS.out.sam_flagstat
+ ch_coverage = SAMTOOLS.out.sam_coverage
QUAST( ch_merged_selected, "03_filtering/quast_filtered" )
ch_quast_report = QUAST.out.report
@@ -97,7 +106,7 @@ workflow STEP_03_ASSEMBLY_FILTER {
emit:
selected_contigs = ch_merged_selected
quast_report = ch_quast_report
- bam = ch_merged_bam
+ bam = ch_final_bam
sam_coverage = ch_coverage
sam_flagstat = ch_flagstat
@@ -158,7 +167,26 @@ process EXTRACT_SINGLE_READS {
script:
"""
- samtools fastq -@ ${task.cpus} -N -1 reads.fastq.gz $bam
+ samtools fastq -@ ${task.cpus} -o reads.fastq.gz $bam
+
+ """
+}
+
+
+
+process MERGE_BAM_FILES {
+ tag "${meta.id}"
+
+ input:
+ tuple val(meta), path(bam1), path(bam2)
+
+ output:
+ tuple val(meta), path("${meta.id}.filtered.bam"), path("${meta.id}.filtered.bam.bai") , emit: bam
+
+ script:
+ """
+ samtools merge -@ ${task.cpus} -o ${meta.id}.filtered.bam $bam1 $bam2
+ samtools index -@ ${task.cpus} ${meta.id}.filtered.bam
"""
}
--
GitLab
From d99f9e2510e7acee9c78c59e74e293058e48a0d7 Mon Sep 17 00:00:00 2001
From: Jean Mainguy <jean.mainguy@inra.fr>
Date: Tue, 25 Oct 2022 16:29:17 +0200
Subject: [PATCH 129/194] change name of input as it can colapse with output
name
---
modules/assembly.nf | 4 ++--
1 file changed, 2 insertions(+), 2 deletions(-)
diff --git a/modules/assembly.nf b/modules/assembly.nf
index 41db2c5..9155270 100644
--- a/modules/assembly.nf
+++ b/modules/assembly.nf
@@ -113,14 +113,14 @@ process RENAME_CONTIGS {
label 'PYTHON'
input:
- tuple val(meta), path(assembly)
+ tuple val(meta), path("${meta.id}.raw.fna")
output:
tuple val(meta), path("${meta.id}.fna"), emit: fna
script:
"""
- rename_contigs.py --sample ${meta.id} --fna_file $assembly --out_fna ${meta.id}.fna -v
+ rename_contigs.py --sample ${meta.id} --fna_file ${meta.id}.raw.fna --out_fna ${meta.id}.fna -v
"""
}
\ No newline at end of file
--
GitLab
From 46c31f5f9af868c710df31b46106747c66658915 Mon Sep 17 00:00:00 2001
From: Jean Mainguy <jean.mainguy@inra.fr>
Date: Tue, 25 Oct 2022 16:34:26 +0200
Subject: [PATCH 130/194] add verbose parameter to filter_contig_per_cpm.py
script
---
modules/filtering_cpm.nf | 2 +-
1 file changed, 1 insertion(+), 1 deletion(-)
diff --git a/modules/filtering_cpm.nf b/modules/filtering_cpm.nf
index 0155034..cdfe8e5 100644
--- a/modules/filtering_cpm.nf
+++ b/modules/filtering_cpm.nf
@@ -12,7 +12,7 @@ process CHUNK_ASSEMBLY_FILTER {
script:
chunk_name = assembly_file.baseName
"""
- filter_contig_per_cpm.py -i ${idxstats} -f ${assembly_file} -c ${min_cpm} -s ${chunk_name}_select_cpm${min_cpm}.fasta -d ${chunk_name}_discard_cpm${min_cpm}.fasta
+ filter_contig_per_cpm.py -v -i ${idxstats} -f ${assembly_file} -c ${min_cpm} -s ${chunk_name}_select_cpm${min_cpm}.fasta -d ${chunk_name}_discard_cpm${min_cpm}.fasta
"""
}
--
GitLab
From 60436117ff467e61b976eff7d525949da7f06278 Mon Sep 17 00:00:00 2001
From: Jean Mainguy <jean.mainguy@inra.fr>
Date: Tue, 25 Oct 2022 16:35:20 +0200
Subject: [PATCH 131/194] publish bam in reads deduplication
---
modules/reads_deduplication.nf | 1 +
1 file changed, 1 insertion(+)
diff --git a/modules/reads_deduplication.nf b/modules/reads_deduplication.nf
index 4731f9b..bce077d 100644
--- a/modules/reads_deduplication.nf
+++ b/modules/reads_deduplication.nf
@@ -2,6 +2,7 @@ process READS_DEDUPLICATION {
tag "${meta.id}"
publishDir "${params.outdir}/02_assembly/deduplicated_reads/", mode: 'copy', pattern: '*.fastq.gz'
publishDir "${params.outdir}/02_assembly/${meta.id}/", mode: 'copy', pattern: '*.fastq.gz'
+ publishDir "${params.outdir}/02_assembly/${meta.id}/", mode: 'copy', pattern: '*.bam*'
input:
tuple val(meta), path(assembly), path(reads)
--
GitLab
From a186a26889578dfaae2fcdca6e24dab471c7f87b Mon Sep 17 00:00:00 2001
From: Jean Mainguy <jean.mainguy@inra.fr>
Date: Tue, 25 Oct 2022 16:41:10 +0200
Subject: [PATCH 132/194] adjust output of S03_FILTERING
---
main.nf | 7 ++++---
1 file changed, 4 insertions(+), 3 deletions(-)
diff --git a/main.nf b/main.nf
index 2c53770..48338b6 100644
--- a/main.nf
+++ b/main.nf
@@ -418,7 +418,7 @@ workflow {
}
if ( !params.stop_at_clean && !params.stop_at_assembly && !params.skip_filtering ) {
- print("IN FILTERING")
+
ch_min_contigs_cpm = Channel.value(params.min_contigs_cpm)
@@ -432,9 +432,10 @@ workflow {
ch_assembly = S03_FILTERING.out.selected_contigs
ch_bam = S03_FILTERING.out.bam
- // ch_sam_coverage = S03_FILTERING.out.coverage
+
+ ch_sam_coverage = S03_FILTERING.out.sam_coverage
ch_quast_after_filter_report = S03_FILTERING.out.quast_report
- // ch_final_assembly_flagstat_report = S03_FILTERING.out.sam_flagstat
+ ch_final_assembly_flagstat_report = S03_FILTERING.out.sam_flagstat
}
--
GitLab
From cb12ad0580f35b6a95c4229f6bdb86aeac85148f Mon Sep 17 00:00:00 2001
From: Jean Mainguy <jean.mainguy@inra.fr>
Date: Tue, 25 Oct 2022 17:33:14 +0200
Subject: [PATCH 133/194] cleaning and restructuration of samtools process
---
main.nf | 11 +--
modules/read_alignment_manipulation.nf | 104 ++++++++++++++++++++++++
modules/read_alignment_metrics.nf | 24 ------
subworkflows/02_assembly.nf | 15 ++--
subworkflows/03_filtering.nf | 105 +++----------------------
subworkflows/05_alignment.nf | 27 -------
subworkflows/08_binning.nf | 8 +-
7 files changed, 125 insertions(+), 169 deletions(-)
create mode 100644 modules/read_alignment_manipulation.nf
delete mode 100644 modules/read_alignment_metrics.nf
diff --git a/main.nf b/main.nf
index 48338b6..cdd2d19 100644
--- a/main.nf
+++ b/main.nf
@@ -453,20 +453,13 @@ workflow {
ch_software_versions_S04 = S04_STRUCTURAL_ANNOT.out.software_versions
}
- // ch_bam = Channel.empty()
ch_diamond_result = Channel.empty()
- // ch_sam_coverage = Channel.empty()
if ( !params.stop_at_clean && !params.stop_at_assembly && !params.stop_at_filtering && !params.stop_at_structural_annot ) {
S05_ALIGNMENT (ch_annotation_faa, ch_diamon_db)
- // ch_bam = S05_ALIGNMENT.out.bam
+
ch_diamond_result = S05_ALIGNMENT.out.m8
- // ch_sam_coverage = S05_ALIGNMENT.out.sam_coverage
- // if (!params.skip_filtering){
- // // when filtering is skip reads vs assembly remain unchanged so no need to send it to multiqc
- // // ch_final_assembly_flagstat_report = S05_ALIGNMENT.out.sam_flagstat
- // }
ch_software_versions_S05 = S05_ALIGNMENT.out.software_versions
}
@@ -486,7 +479,7 @@ workflow {
}
if ( !params.stop_at_clean && !params.stop_at_assembly && !params.stop_at_filtering && !params.stop_at_structural_annot && !params.skip_binning ) {
- S08_BINNING( ch_reads, ch_annotation_fna, ch_bam, ch_gtbdtk_db, ch_quast )
+ S08_BINNING( ch_reads, ch_assembly, ch_bam, ch_gtbdtk_db, ch_quast )
ch_bins_abundances_report = S08_BINNING.out.bins_abundances_report
ch_bins_stats_report = S08_BINNING.out.bins_stats_report
diff --git a/modules/read_alignment_manipulation.nf b/modules/read_alignment_manipulation.nf
new file mode 100644
index 0000000..ca0fe5d
--- /dev/null
+++ b/modules/read_alignment_manipulation.nf
@@ -0,0 +1,104 @@
+process GET_ALIGNMENT_METRICS {
+ tag "${meta.id}"
+ publishDir "${params.outdir}/$publishDir_path/${meta.id}", mode: 'copy'
+
+ input:
+ tuple val(meta), path(bam), path(bai)
+ val(publishDir_path)
+
+ output:
+ tuple val(meta), path("${meta.id}.coverage.tsv"), emit: sam_coverage
+ tuple val(meta), path("${meta.id}.idxstats"), emit: sam_idxstat
+ path "${meta.id}.flagstat", emit: sam_flagstat
+ path "${meta.id}*"
+ path "v_samtools.txt", emit: v_samtools
+
+ script:
+ """
+ samtools flagstat -@ ${task.cpus} ${bam} > ${meta.id}.flagstat
+ samtools coverage ${bam} > ${meta.id}.coverage.tsv
+ samtools idxstats ${bam} > ${meta.id}.idxstats
+
+ samtools --version &> v_samtools.txt
+ """
+}
+
+
+process FILTER_BAM {
+ tag "${meta.id}"
+
+ input:
+ tuple val(meta), path(discarded_contigs), path(bam), path(bai)
+
+ output:
+ tuple val(meta), path("selected_contigs.bam"), emit: selected_contigs_bam
+ tuple val(meta), path("discarded_contigs.bam"), emit: discarded_contigs_bam
+
+
+ script:
+ """
+
+ samtools faidx $discarded_contigs
+ awk 'BEGIN {FS="\\t"}; {print \$1 FS "0" FS \$2}' ${discarded_contigs}.fai > discarded_contigs.bed
+
+
+ samtools view -@ ${task.cpus} -bL discarded_contigs.bed -U selected_contigs.bam $bam -o discarded_contigs.bam
+
+
+ """
+}
+
+process EXTRACT_PAIRED_READS {
+ tag "${meta.id}"
+
+ input:
+ tuple val(meta), path(bam)
+
+ output:
+ tuple val(meta), path("reads*.fastq.gz"), emit: reads
+
+
+ script:
+ """
+
+ samtools fastq -@ ${task.cpus} -N -1 reads_R1.fastq.gz -2 reads_R2.fastq.gz $bam
+
+ """
+}
+
+process EXTRACT_SINGLE_READS {
+ tag "${meta.id}"
+
+ input:
+ tuple val(meta), path(bam)
+
+ output:
+ tuple val(meta), path("reads.fastq.gz"), emit: reads
+
+ script:
+ """
+
+ samtools fastq -@ ${task.cpus} -o reads.fastq.gz $bam
+
+ """
+}
+
+
+
+process MERGE_BAM_FILES {
+ tag "${meta.id}"
+
+ input:
+ tuple val(meta), path(bam1), path(bam2)
+
+ output:
+ tuple val(meta), path("${meta.id}.filtered.bam"), path("${meta.id}.filtered.bam.bai") , emit: bam
+
+ script:
+ """
+ samtools merge -@ ${task.cpus} -o ${meta.id}.filtered.bam $bam1 $bam2
+ samtools index -@ ${task.cpus} ${meta.id}.filtered.bam
+
+ """
+}
+
diff --git a/modules/read_alignment_metrics.nf b/modules/read_alignment_metrics.nf
deleted file mode 100644
index ee1bd54..0000000
--- a/modules/read_alignment_metrics.nf
+++ /dev/null
@@ -1,24 +0,0 @@
-process SAMTOOLS {
- tag "${meta.id}"
- publishDir "${params.outdir}/$publishDir_path/${meta.id}", mode: 'copy'
-
- input:
- tuple val(meta), path(bam), path(bai)
- val(publishDir_path)
-
- output:
- tuple val(meta), path("${meta.id}.coverage.tsv"), emit: sam_coverage
- tuple val(meta), path("${meta.id}.idxstats"), emit: sam_idxstat
- path "${meta.id}.flagstat", emit: sam_flagstat
- path "${meta.id}*"
- path "v_samtools.txt", emit: v_samtools
-
- script:
- """
- samtools flagstat -@ ${task.cpus} ${bam} > ${meta.id}.flagstat
- samtools coverage ${bam} > ${meta.id}.coverage.tsv
- samtools idxstats ${bam} > ${meta.id}.idxstats
-
- samtools --version &> v_samtools.txt
- """
-}
\ No newline at end of file
diff --git a/subworkflows/02_assembly.nf b/subworkflows/02_assembly.nf
index ee5a207..fe90d44 100644
--- a/subworkflows/02_assembly.nf
+++ b/subworkflows/02_assembly.nf
@@ -2,7 +2,7 @@ include { METASPADES; MEGAHIT; HIFIASM_META; METAFLYE; RENAME_CONTIGS } from '..
include { QUAST as ASSEMBLY_QUAST} from '../modules/metaquast'
include { READS_DEDUPLICATION } from '../modules/reads_deduplication'
include { MINIMAP2 } from '../modules/read_alignment'
-include { SAMTOOLS } from '../modules/read_alignment_metrics'
+include { GET_ALIGNMENT_METRICS } from '../modules/read_alignment_manipulation'
workflow STEP_02_ASSEMBLY {
take:
@@ -67,22 +67,19 @@ workflow STEP_02_ASSEMBLY {
ch_bwa_mem_v = READS_DEDUPLICATION.out.v_bwa_mem2
} else {
-
- if (!params.skip_filtering) {
MINIMAP2(ch_contigs_and_reads,"02_assembly")
ch_bam = MINIMAP2.out.bam
ch_minimap2_v = MINIMAP2.out.v_minimap2
- }
}
- SAMTOOLS(ch_bam, "02_assembly")
+ GET_ALIGNMENT_METRICS(ch_bam, "02_assembly")
- ch_idxstats = SAMTOOLS.out.sam_idxstat
- ch_flagstat = SAMTOOLS.out.sam_flagstat
- ch_coverage = SAMTOOLS.out.sam_coverage
- ch_samtools_v = SAMTOOLS.out.v_samtools
+ ch_idxstats = GET_ALIGNMENT_METRICS.out.sam_idxstat
+ ch_flagstat = GET_ALIGNMENT_METRICS.out.sam_flagstat
+ ch_coverage = GET_ALIGNMENT_METRICS.out.sam_coverage
+ ch_samtools_v = GET_ALIGNMENT_METRICS.out.v_samtools
ch_software_versions = ch_assembler_v.first().mix( ch_quast_v.first(),
ch_bwa_mem_v.first(),
diff --git a/subworkflows/03_filtering.nf b/subworkflows/03_filtering.nf
index 97399ef..be38cd6 100644
--- a/subworkflows/03_filtering.nf
+++ b/subworkflows/03_filtering.nf
@@ -1,7 +1,7 @@
include { CHUNK_ASSEMBLY_FILTER; MERGE_ASSEMBLY_FILTER} from '../modules/filtering_cpm.nf'
include { QUAST } from '../modules/metaquast'
include { MINIMAP2; BWA_MEM } from '../modules/read_alignment'
-include { SAMTOOLS } from '../modules/read_alignment_metrics'
+include { GET_ALIGNMENT_METRICS; FILTER_BAM; EXTRACT_SINGLE_READS; EXTRACT_PAIRED_READS; MERGE_BAM_FILES} from '../modules/read_alignment_manipulation'
workflow STEP_03_ASSEMBLY_FILTER {
take:
@@ -41,6 +41,9 @@ workflow STEP_03_ASSEMBLY_FILTER {
ch_merged_selected = MERGE_ASSEMBLY_FILTER.out.merged_selected
discarded_contigs = MERGE_ASSEMBLY_FILTER.out.merged_discarded
+
+ // Differentiate sample with and without discarded_contigs
+ // samples with no discarded_contigs are not going to be processed to process
discarded_contigs_category = discarded_contigs.branch{
without: it[1].isEmpty()
with: !it[1].isEmpty()
@@ -48,15 +51,9 @@ workflow STEP_03_ASSEMBLY_FILTER {
samples_without_discarded_contigs = discarded_contigs_category.without.map{ it -> it[0]}
- samples_without_discarded_contigs.view{"samples WITHOUT DISCARDED CONTIGS: $it"}
ch_bam_of_sample_without_discarded_contigs = samples_without_discarded_contigs.join(bam)
- ch_bam_of_sample_without_discarded_contigs.view{"samples WITHOUT DISCARDED CONTIGS AND bam: $it"}
-
- discarded_contigs_category.without.view { "WITHOUT::::::::: > $it" }
- discarded_contigs_category.with.view { "WITH::::::::: > $it" }
ch_discarded_contigs_and_bam = discarded_contigs_category.with.join(bam)
- ch_discarded_contigs_and_bam.view()
FILTER_BAM(ch_discarded_contigs_and_bam)
@@ -75,30 +72,24 @@ workflow STEP_03_ASSEMBLY_FILTER {
} else {
ch_discarded_reads = EXTRACT_SINGLE_READS(ch_discarded_bam)
ch_contigs_and_discarded_reads = ch_merged_selected.join(ch_discarded_reads)
- MINIMAP2(ch_contigs_and_reads, "03_filtering")
+ MINIMAP2(ch_contigs_and_discarded_reads, "03_filtering")
ch_bam_with_discarded_reads = MINIMAP2.out.bam
}
- // remove bai from ch_bam_with_discarded_reads
+ // remove bai from ch_bam_with_discarded_reads and add bam of selected contigs
ch_bam_to_merge = ch_bam_with_discarded_reads.map{ it -> [it[0], it[1]]}.join(ch_selected_bam)
-
-
-
-
ch_merged_bam = MERGE_BAM_FILES(ch_bam_to_merge)
ch_final_bam = ch_bam_of_sample_without_discarded_contigs.mix(ch_merged_bam)
- ch_final_bam.view { "FINAL BAMMMM::::::::: > $it" }
-
- SAMTOOLS(ch_final_bam, '03_filtering/')
+ GET_ALIGNMENT_METRICS(ch_final_bam, '03_filtering/')
- ch_flagstat = SAMTOOLS.out.sam_flagstat
- ch_coverage = SAMTOOLS.out.sam_coverage
+ ch_flagstat = GET_ALIGNMENT_METRICS.out.sam_flagstat
+ ch_coverage = GET_ALIGNMENT_METRICS.out.sam_coverage
QUAST( ch_merged_selected, "03_filtering/quast_filtered" )
ch_quast_report = QUAST.out.report
@@ -112,81 +103,3 @@ workflow STEP_03_ASSEMBLY_FILTER {
}
-
-process FILTER_BAM {
- tag "${meta.id}"
-
- input:
- tuple val(meta), path(discarded_contigs), path(bam), path(bai)
-
- output:
- tuple val(meta), path("selected_contigs.bam"), emit: selected_contigs_bam
- tuple val(meta), path("discarded_contigs.bam"), emit: discarded_contigs_bam
-
-
- script:
- """
-
- samtools faidx $discarded_contigs
- awk 'BEGIN {FS="\\t"}; {print \$1 FS "0" FS \$2}' ${discarded_contigs}.fai > discarded_contigs.bed
-
-
- samtools view -@ ${task.cpus} -bL discarded_contigs.bed -U selected_contigs.bam $bam -o discarded_contigs.bam
-
-
- """
-}
-
-process EXTRACT_PAIRED_READS {
- tag "${meta.id}"
-
- input:
- tuple val(meta), path(bam) //, path(bai)
-
- output:
- tuple val(meta), path("reads*.fastq.gz"), emit: reads
-
-
- script:
- """
-
- samtools fastq -@ ${task.cpus} -N -1 reads_R1.fastq.gz -2 reads_R2.fastq.gz $bam
-
- """
-}
-
-process EXTRACT_SINGLE_READS {
- tag "${meta.id}"
-
- input:
- tuple val(meta), path(bam), path(bai)
-
- output:
- tuple val(meta), path("reads.fastq.gz"), emit: reads
-
- script:
- """
-
- samtools fastq -@ ${task.cpus} -o reads.fastq.gz $bam
-
- """
-}
-
-
-
-process MERGE_BAM_FILES {
- tag "${meta.id}"
-
- input:
- tuple val(meta), path(bam1), path(bam2)
-
- output:
- tuple val(meta), path("${meta.id}.filtered.bam"), path("${meta.id}.filtered.bam.bai") , emit: bam
-
- script:
- """
- samtools merge -@ ${task.cpus} -o ${meta.id}.filtered.bam $bam1 $bam2
- samtools index -@ ${task.cpus} ${meta.id}.filtered.bam
-
- """
-}
diff --git a/subworkflows/05_alignment.nf b/subworkflows/05_alignment.nf
index 191d20f..9902230 100644
--- a/subworkflows/05_alignment.nf
+++ b/subworkflows/05_alignment.nf
@@ -1,37 +1,13 @@
-include { MINIMAP2; BWA_MEM } from '../modules/read_alignment'
-include { SAMTOOLS } from '../modules/read_alignment_metrics'
include { DIAMOND } from '../modules/diamond'
workflow STEP_05_ALIGNMENT {
take:
- // contigs_and_reads
prokka_faa
diamond
main:
- // ch_bwa_mem_v = Channel.empty()
- // ch_minimap2_v = Channel.empty()
- // ch_samtools_v = Channel.empty()
ch_diamond_v = Channel.empty()
- // publishDir = "05_alignment/05_1_reads_alignment_on_contigs"
- // if (params.type == 'SR') {
- // BWA_MEM(contigs_and_reads, publishDir)
- // ch_bam = BWA_MEM.out.bam
-
- // ch_bwa_mem_v = BWA_MEM.out.v_bwa_mem2
- // ch_samtools_v = BWA_MEM.out.v_samtools
- // } else {
- // MINIMAP2(contigs_and_reads, publishDir)
- // ch_bam = MINIMAP2.out.bam
-
- // ch_minimap2_v = MINIMAP2.out.v_minimap2
- // ch_samtools_v = MINIMAP2.out.v_samtools
- // }
-
- // SAMTOOLS(ch_bam, publishDir)
- // ch_sam_coverage = SAMTOOLS.out.sam_coverage
- // ch_sam_flagstat = SAMTOOLS.out.sam_flagstat
ch_m8 =Channel.empty()
if (!params.skip_func_annot || !params.skip_taxo_affi){
@@ -43,9 +19,6 @@ workflow STEP_05_ALIGNMENT {
ch_diamond_v = DIAMOND.out.v_diamond
}
emit:
- // bam = ch_bam
m8 = ch_m8
- // sam_coverage = ch_sam_coverage
software_versions = ch_diamond_v.first()
- // sam_flagstat = ch_sam_flagstat
}
diff --git a/subworkflows/08_binning.nf b/subworkflows/08_binning.nf
index 97df1a6..59b9c7b 100644
--- a/subworkflows/08_binning.nf
+++ b/subworkflows/08_binning.nf
@@ -2,7 +2,7 @@ include { GENERATE_DEPTH_FILES; METABAT2; MAXBIN2; CONCOCT; METAWRAP_REFINMENT;
include { CHECKM2 } from '../modules/checkm2'
include { DREP } from '../modules/drep'
include { BWA_MEM;BWA_MEM as BWA_MEM_CROSS_ALIGNMENT; MINIMAP2; MINIMAP2 as MINIMAP2_CROSS_ALIGNMENT } from '../modules/read_alignment'
-include { SAMTOOLS } from '../modules/read_alignment_metrics'
+include { GET_ALIGNMENT_METRICS } from '../modules/read_alignment_manipulation'
include { GTDBTK } from '../modules/gtdbtk'
include { GENOMES_ABUNDANCES_PER_SAMPLE; ADD_QUAST_INFO_TO_BINS; BINS_STATS_TO_MUTLIQC_FORMAT} from '../modules/sum_up_bins_informations'
@@ -213,11 +213,11 @@ workflow STEP_08_BINNING {
ch_collect_coverages = Channel.empty()
ch_collect_flagstats = Channel.empty()
- SAMTOOLS(ch_bam, "08_binning/08_4_mapping_on_final_bins/stats")
+ GET_ALIGNMENT_METRICS(ch_bam, "08_binning/08_4_mapping_on_final_bins/stats")
- ch_collect_coverages = SAMTOOLS.out.sam_coverage.map {id, file -> file}
+ ch_collect_coverages = GET_ALIGNMENT_METRICS.out.sam_coverage.map {id, file -> file}
.collect()
- ch_collect_flagstats = SAMTOOLS.out.sam_flagstat.collect()
+ ch_collect_flagstats = GET_ALIGNMENT_METRICS.out.sam_flagstat.collect()
GENOMES_ABUNDANCES_PER_SAMPLE(ch_collect_coverages, ch_collect_flagstats, \
ch_bins_drep, DREP.out.output_drep_stats, GTDBTK.out.gtdbtk_affiliations_predictions,
--
GitLab
From ae9017be411d1df521afb65d55ffdd9d3f6adafa Mon Sep 17 00:00:00 2001
From: Jean Mainguy <jean.mainguy@inra.fr>
Date: Tue, 25 Oct 2022 17:33:49 +0200
Subject: [PATCH 134/194] comment prokka warning
---
docs/usage.md | 4 ++--
1 file changed, 2 insertions(+), 2 deletions(-)
diff --git a/docs/usage.md b/docs/usage.md
index 672fa3a..d0b1a4c 100644
--- a/docs/usage.md
+++ b/docs/usage.md
@@ -300,7 +300,7 @@ No parameter available for this substep.
No parameters.
**WARNING 6:** `S04_STRUCTURAL_ANNOT` step depends on `S01_CLEAN_QC`, `S02_ASSEMBLY` and `S03_FILTERING` steps (if you use it). You need to use the mandatory files of these four steps to run `S04_STRUCTURAL_ANNOT`. See [II. Input files](usage.md#ii-input-files) and **WARNINGS 1 to 6**.
-
+<!--
**WARNING 7:** if you haven't previously done `S03_FILTERING`, calculation time of `S04_STRUCTURAL_ANNOT` can be important. Some cluster queues have defined calculation time, you need to adapt the queue you use to your data.
> For example, if you are on [genologin cluster](http://bioinfo.genotoul.fr/) and you haven't done the `S03_FILTERING` step, you can write a `nextflow.config` file in your working directory containing these lines:
> ```bash
@@ -308,7 +308,7 @@ No parameters.
> queue = 'unlimitq'
> }
> ```
-> This will launch the `Prokka` command line of step `04_STRUCTURAL_ANNOT` on a calculation queue (`unlimitq`) where the job can last more than 4 days (which is not the case for the usual `workq` queue).
+> This will launch the `Prokka` command line of step `04_STRUCTURAL_ANNOT` on a calculation queue (`unlimitq`) where the job can last more than 4 days (which is not the case for the usual `workq` queue). -->
#### **`S05_ALIGNMENT` step:**
--
GitLab
From fba3aa0cd4f4b460ae8cb680b3297c264768e6b7 Mon Sep 17 00:00:00 2001
From: Jean Mainguy <jean.mainguy@inra.fr>
Date: Wed, 26 Oct 2022 12:55:33 +0200
Subject: [PATCH 135/194] correct unsolved conflict
---
bin/merge_annotations.py | 13 -------------
1 file changed, 13 deletions(-)
diff --git a/bin/merge_annotations.py b/bin/merge_annotations.py
index 8aa0ea3..d18ebd0 100755
--- a/bin/merge_annotations.py
+++ b/bin/merge_annotations.py
@@ -206,19 +206,6 @@ def writing_features_to_gff_ffn_faa(annotations_per_contig, out_gff, fna_file, o
-<<<<<<< HEAD
- start_zero, end_zero = int(feature['start'])-1, int(feature['end'])
-
- if feature['strand'] == "+":
- feature_seq = contig_fa[contig][start-1:end].seq
- else:
- # minus strand: reverse complement the sequence
- feature_seq = contig_fa[contig][start-1:end].antisense
-
- fh_ffn.write(f">{new_id} {start}-{end} {tag_name}\n{feature_seq}\n")
-
-=======
->>>>>>> 734ac13e66692f25d221fb22bd2204737a2875f1
def main():
--
GitLab
From de39906a8397c9162f46d213c6976a77b1b76656 Mon Sep 17 00:00:00 2001
From: Jean Mainguy <jean.mainguy@inra.fr>
Date: Thu, 27 Oct 2022 16:22:31 +0200
Subject: [PATCH 136/194] add the possibility to have a gzip reference genome
#202
---
bin/db_versions.py | 6 +++++-
1 file changed, 5 insertions(+), 1 deletion(-)
diff --git a/bin/db_versions.py b/bin/db_versions.py
index becb567..7179672 100755
--- a/bin/db_versions.py
+++ b/bin/db_versions.py
@@ -3,6 +3,7 @@
import argparse
import subprocess
import os.path
+import gzip
def info_db(file):
name=file.split()[0]
@@ -22,8 +23,11 @@ def info_db(file):
def get_genome_seq_count(genome_path):
+
seq_count = 0
- with open(genome_path,"r") as fh:
+
+ proper_open = gzip.open if genome_path.endswith('.gz') else open
+ with proper_open(genome_path,"r") as fh:
for l in fh:
if l.startswith('>'):
seq_count += 1
--
GitLab
From fe4a7ecf18f58da660c2c6a2714be46c08ae29d9 Mon Sep 17 00:00:00 2001
From: Jean Mainguy <jean.mainguy@inra.fr>
Date: Fri, 28 Oct 2022 11:12:00 +0200
Subject: [PATCH 137/194] add proper opening to read gzip file #202
---
bin/db_versions.py | 2 +-
1 file changed, 1 insertion(+), 1 deletion(-)
diff --git a/bin/db_versions.py b/bin/db_versions.py
index 7179672..3088ea3 100755
--- a/bin/db_versions.py
+++ b/bin/db_versions.py
@@ -27,7 +27,7 @@ def get_genome_seq_count(genome_path):
seq_count = 0
proper_open = gzip.open if genome_path.endswith('.gz') else open
- with proper_open(genome_path,"r") as fh:
+ with proper_open(genome_path,"rt") as fh:
for l in fh:
if l.startswith('>'):
seq_count += 1
--
GitLab
From 157d2189915f0cf1b36caf2514641ec25ad0030b Mon Sep 17 00:00:00 2001
From: Jean Mainguy <jean.mainguy@inra.fr>
Date: Wed, 2 Nov 2022 14:01:24 +0100
Subject: [PATCH 138/194] add gene line gff file
---
bin/merge_annotations.py | 22 ++++++++++++++++------
1 file changed, 16 insertions(+), 6 deletions(-)
diff --git a/bin/merge_annotations.py b/bin/merge_annotations.py
index d18ebd0..afa038a 100755
--- a/bin/merge_annotations.py
+++ b/bin/merge_annotations.py
@@ -139,10 +139,10 @@ def merging_cds_and_rna(cds_per_contig, contig2rnas):
logging.info(f'{contig} has only rna annotation')
yield contig, rnas
-def add_new_ID_tag(gff_attributes, new_id):
+def add_new_ID_tag_and_Parent(gff_attributes, new_id, parent_id):
- gff_attributes_no_id = ';'.join([attr for attr in gff_attributes.split(';') if attr.split('=')[0] != 'ID'])
- return f"ID={new_id};{gff_attributes_no_id}"
+ gff_attributes_no_id = ';'.join([attr for attr in gff_attributes.split(';') if attr.split('=')[0] not in ['ID', 'Parent']])
+ return f"ID={new_id};Parent={parent_id};{gff_attributes_no_id}"
def get_tag_value(gff_attribute, tag):
@@ -168,6 +168,7 @@ def writing_features_to_gff_ffn_faa(annotations_per_contig, out_gff, fna_file, o
for feature in sorted(features, key=lambda x: int(x['start'])):
gene_count[feature['feature']] += 1
new_id = f"{feature['seqname']}.{feature['feature']}_{gene_count[feature['feature']]}"
+ parent_id = f"{new_id}_gene"
start, end = int(feature['start']), int(feature['end'])
@@ -183,11 +184,20 @@ def writing_features_to_gff_ffn_faa(annotations_per_contig, out_gff, fna_file, o
# Write gff and ffn
- feature['attribute'] = add_new_ID_tag(feature['attribute'], new_id)
+ feature['attribute'] = add_new_ID_tag_and_Parent(feature['attribute'], new_id, parent_id)
+
+ # gff line with CDS/tRNA or rRNA in feature column
+ feature_gff_line = '\t'.join(feature.values())
+
+ # gff line with gene in feature column
+ feature['feature'] = 'gene'
+ feature['attribute'] = f'ID={parent_id};locus_tag={new_id}'
+ gene_gff_line = '\t'.join(feature.values())
+
+ fh_gff.write(f'{gene_gff_line}\n{feature_gff_line}\n')
tag_name = get_tag_value(feature['attribute'], 'Name')
- fh_gff.write('\t'.join(feature.values())+"\n")
-
+
if feature['strand'] == "+":
feature_seq = contig_fa[contig][start-1:end].seq
else:
--
GitLab
From 160e6e786ce6b17a2f561c23a4f89c4ca6e7ac62 Mon Sep 17 00:00:00 2001
From: Jean Mainguy <jean.mainguy@inra.fr>
Date: Wed, 2 Nov 2022 14:17:15 +0100
Subject: [PATCH 139/194] add old name as description of renamed sequenced to
keep it
---
bin/rename_contigs.py | 4 ++--
1 file changed, 2 insertions(+), 2 deletions(-)
diff --git a/bin/rename_contigs.py b/bin/rename_contigs.py
index 0734c9c..264d9cf 100755
--- a/bin/rename_contigs.py
+++ b/bin/rename_contigs.py
@@ -64,8 +64,8 @@ def main():
logging.info(f'Writting renamed fasta file in {out_fna}')
with open(out_fna, "w") as fh_fna:
- for i, (_, seq) in enumerate(pyfastx.Fasta(fna_file, build_index=False)):
- fh_fna.write(f'>{sample}_c{i+1}\n{seq}\n')
+ for i, (name, seq) in enumerate(pyfastx.Fasta(fna_file, build_index=False)):
+ fh_fna.write(f'>{sample}_c{i+1} {name}\n{seq}\n')
if __name__ == '__main__':
main()
\ No newline at end of file
--
GitLab
From f5c608a55e67e7c668734a78a23a5a9491f5bda1 Mon Sep 17 00:00:00 2001
From: Jean Mainguy <jean.mainguy@inra.fr>
Date: Wed, 2 Nov 2022 14:27:34 +0100
Subject: [PATCH 140/194] add flag skip_alignment to skip step 05
---
main.nf | 7 ++++---
1 file changed, 4 insertions(+), 3 deletions(-)
diff --git a/main.nf b/main.nf
index cdd2d19..8339cab 100644
--- a/main.nf
+++ b/main.nf
@@ -77,6 +77,7 @@ include { MERGE_FASTQ } from './modules/merge_fastq.nf'
S05_ALIGNMENT options:
--diamond_bank Path to diamond bank used to align protein sequence of genes: "PATH/bank.dmnd".
This bank must be previously built with diamond makedb.
+ --skip_alignment Skip this step
S06_FUNC_ANNOT options:
--skip_func_annot Skip this step
@@ -455,7 +456,7 @@ workflow {
ch_diamond_result = Channel.empty()
- if ( !params.stop_at_clean && !params.stop_at_assembly && !params.stop_at_filtering && !params.stop_at_structural_annot ) {
+ if ( !params.stop_at_clean && !params.stop_at_assembly && !params.stop_at_filtering && !params.stop_at_structural_annot && !params.skip_alignment) {
S05_ALIGNMENT (ch_annotation_faa, ch_diamon_db)
ch_diamond_result = S05_ALIGNMENT.out.m8
@@ -465,14 +466,14 @@ workflow {
ch_quant_report = Channel.empty()
ch_v_eggnogmapper = Channel.empty()
- if ( !params.stop_at_clean && !params.stop_at_assembly && !params.stop_at_filtering && !params.stop_at_structural_annot && !params.skip_func_annot ) {
+ if ( !params.stop_at_clean && !params.stop_at_assembly && !params.stop_at_filtering && !params.stop_at_structural_annot && !params.skip_func_annot && !params.skip_alignment) {
S06_FUNC_ANNOT ( ch_annotation_ffn, ch_annotation_faa, ch_annotation_gff, ch_bam, ch_diamond_result, ch_eggnog_db )
ch_quant_report = S06_FUNC_ANNOT.out.quant_report
ch_software_versions_S06 = S06_FUNC_ANNOT.out.software_versions
}
- if ( !params.stop_at_clean && !params.stop_at_assembly && !params.stop_at_filtering && !params.stop_at_structural_annot && !params.skip_taxo_affi ) {
+ if ( !params.stop_at_clean && !params.stop_at_assembly && !params.stop_at_filtering && !params.stop_at_structural_annot && !params.skip_taxo_affi && !params.skip_alignment) {
S07_TAXO_AFFI ( ch_taxonomy, ch_diamond_result, ch_sam_coverage)
ch_software_versions_S07 = S07_TAXO_AFFI.out.software_versions
--
GitLab
From e4479d10ea3e129adc1c53e6a1e3f17a0b500796 Mon Sep 17 00:00:00 2001
From: Jean Mainguy <jean.mainguy@inra.fr>
Date: Fri, 4 Nov 2022 15:58:10 +0100
Subject: [PATCH 141/194] Improve fasta parsing
---
bin/Bins_per_sample_summarize.py | 5 +++--
1 file changed, 3 insertions(+), 2 deletions(-)
diff --git a/bin/Bins_per_sample_summarize.py b/bin/Bins_per_sample_summarize.py
index fce7b72..89efa2b 100755
--- a/bin/Bins_per_sample_summarize.py
+++ b/bin/Bins_per_sample_summarize.py
@@ -45,13 +45,14 @@ def bins_contigs_compositions(folder):
contigs_to_bins = dict()
list_bins = list()
for fi in os.listdir(folder):
- with open(folder + "/" + fi,'r') as bin_file:
+ bin_fasta_file = os.path.join(folder, fi)
+ with open(bin_fasta_file) as bin_file:
bin_name = fi.rsplit('.', maxsplit=1)[0]
if bin_name not in list_bins:
list_bins.append(bin_name)
for line in bin_file:
if line.startswith('>'):
- contig_name = line.strip().lstrip('>')
+ contig_name = line.split()[0].strip().lstrip('>')
contigs_to_bins[contig_name] = bin_name
return contigs_to_bins, list_bins
--
GitLab
From 391de61a607efd8fc247fc5934059a85b4108c6d Mon Sep 17 00:00:00 2001
From: Jean Mainguy <jean.mainguy@inra.fr>
Date: Fri, 4 Nov 2022 15:58:41 +0100
Subject: [PATCH 142/194] add meta to quast report because it is needed in
binning step
---
main.nf | 9 +++++++--
modules/metaquast.nf | 2 +-
2 files changed, 8 insertions(+), 3 deletions(-)
diff --git a/main.nf b/main.nf
index 8339cab..418f850 100644
--- a/main.nf
+++ b/main.nf
@@ -414,7 +414,9 @@ workflow {
ch_idxstats = S02_ASSEMBLY.out.idxstats
ch_unfilter_assembly_flagstat_report = S02_ASSEMBLY.out.flagstat
- ch_quast_before_filter_report = S02_ASSEMBLY.out.assembly_report
+ ch_quast = S02_ASSEMBLY.out.assembly_report
+ ch_quast_before_filter_report = ch_quast.map{ meta, quast_report -> quast_report }
+
ch_software_versions_S02 = S02_ASSEMBLY.out.software_versions
}
@@ -435,7 +437,10 @@ workflow {
ch_bam = S03_FILTERING.out.bam
ch_sam_coverage = S03_FILTERING.out.sam_coverage
- ch_quast_after_filter_report = S03_FILTERING.out.quast_report
+
+ ch_quast = S03_FILTERING.out.quast_report
+ ch_quast_after_filter_report = ch_quast.map{ meta, quast_report -> quast_report }
+
ch_final_assembly_flagstat_report = S03_FILTERING.out.sam_flagstat
}
diff --git a/modules/metaquast.nf b/modules/metaquast.nf
index dee90d5..dbaa508 100644
--- a/modules/metaquast.nf
+++ b/modules/metaquast.nf
@@ -9,7 +9,7 @@ process QUAST {
output:
path "${outdir}/${meta.id}/*", emit: all
- path "${outdir}/${meta.id}/report.tsv", emit: report
+ tuple val(meta), path ("${outdir}/${meta.id}/report.tsv"), emit: report
path "v_quast.txt", emit: v_quast
script:
--
GitLab
From d16b0f1ea95dfc8c056e393d55050a944e1aecf2 Mon Sep 17 00:00:00 2001
From: Jean Mainguy <jean.mainguy@inra.fr>
Date: Mon, 7 Nov 2022 13:02:04 +0100
Subject: [PATCH 143/194] add skip_alignment in config
---
nextflow.config | 3 +++
1 file changed, 3 insertions(+)
diff --git a/nextflow.config b/nextflow.config
index 9e86698..1516f4c 100644
--- a/nextflow.config
+++ b/nextflow.config
@@ -43,6 +43,9 @@ params {
// Step
stop_at_structural_annot = false
+ // Optional step
+ skip_alignment = false
+
// Optional step
skip_func_annot = false
--
GitLab
From 61e8be3c6045abfa7c92b7e995147ab62b1b5f74 Mon Sep 17 00:00:00 2001
From: Jean Mainguy <jean.mainguy@inra.fr>
Date: Mon, 7 Nov 2022 15:45:40 +0100
Subject: [PATCH 144/194] remove skip_alignment flag
---
main.nf | 9 ++++-----
subworkflows/05_alignment.nf | 21 +++++++++++----------
2 files changed, 15 insertions(+), 15 deletions(-)
diff --git a/main.nf b/main.nf
index 418f850..5d5d849 100644
--- a/main.nf
+++ b/main.nf
@@ -77,7 +77,6 @@ include { MERGE_FASTQ } from './modules/merge_fastq.nf'
S05_ALIGNMENT options:
--diamond_bank Path to diamond bank used to align protein sequence of genes: "PATH/bank.dmnd".
This bank must be previously built with diamond makedb.
- --skip_alignment Skip this step
S06_FUNC_ANNOT options:
--skip_func_annot Skip this step
@@ -461,24 +460,24 @@ workflow {
ch_diamond_result = Channel.empty()
- if ( !params.stop_at_clean && !params.stop_at_assembly && !params.stop_at_filtering && !params.stop_at_structural_annot && !params.skip_alignment) {
+ if ( !params.stop_at_clean && !params.stop_at_assembly && !params.stop_at_filtering && !params.stop_at_structural_annot && (!params.skip_func_annot || !params.skip_taxo_affi)) {
S05_ALIGNMENT (ch_annotation_faa, ch_diamon_db)
- ch_diamond_result = S05_ALIGNMENT.out.m8
+ ch_diamond_result = S05_ALIGNMENT.out.diamond_result
ch_software_versions_S05 = S05_ALIGNMENT.out.software_versions
}
ch_quant_report = Channel.empty()
ch_v_eggnogmapper = Channel.empty()
- if ( !params.stop_at_clean && !params.stop_at_assembly && !params.stop_at_filtering && !params.stop_at_structural_annot && !params.skip_func_annot && !params.skip_alignment) {
+ if ( !params.stop_at_clean && !params.stop_at_assembly && !params.stop_at_filtering && !params.stop_at_structural_annot && !params.skip_func_annot) {
S06_FUNC_ANNOT ( ch_annotation_ffn, ch_annotation_faa, ch_annotation_gff, ch_bam, ch_diamond_result, ch_eggnog_db )
ch_quant_report = S06_FUNC_ANNOT.out.quant_report
ch_software_versions_S06 = S06_FUNC_ANNOT.out.software_versions
}
- if ( !params.stop_at_clean && !params.stop_at_assembly && !params.stop_at_filtering && !params.stop_at_structural_annot && !params.skip_taxo_affi && !params.skip_alignment) {
+ if ( !params.stop_at_clean && !params.stop_at_assembly && !params.stop_at_filtering && !params.stop_at_structural_annot && !params.skip_taxo_affi) {
S07_TAXO_AFFI ( ch_taxonomy, ch_diamond_result, ch_sam_coverage)
ch_software_versions_S07 = S07_TAXO_AFFI.out.software_versions
diff --git a/subworkflows/05_alignment.nf b/subworkflows/05_alignment.nf
index 9902230..9017d47 100644
--- a/subworkflows/05_alignment.nf
+++ b/subworkflows/05_alignment.nf
@@ -9,16 +9,17 @@ workflow STEP_05_ALIGNMENT {
ch_diamond_v = Channel.empty()
- ch_m8 =Channel.empty()
- if (!params.skip_func_annot || !params.skip_taxo_affi){
- DIAMOND (
- prokka_faa,
- diamond
- )
- ch_m8 = DIAMOND.out.m8
- ch_diamond_v = DIAMOND.out.v_diamond
- }
+ ch_diamond_result =Channel.empty()
+
+ DIAMOND (
+ prokka_faa,
+ diamond,
+ )
+
+ ch_diamond_result = DIAMOND.out.m8
+ ch_diamond_v = DIAMOND.out.v_diamond
+
emit:
- m8 = ch_m8
+ diamond_result = ch_diamond_result
software_versions = ch_diamond_v.first()
}
--
GitLab
From 242ed941406e2f1811554dbc633879075a429f72 Mon Sep 17 00:00:00 2001
From: VIENNE MAINA <maina.vienne@inrae.fr>
Date: Tue, 8 Nov 2022 10:45:26 +0100
Subject: [PATCH 145/194] refactoring
---
bin/{aln2taxaffi.py => aln_to_tax_affi.py} | 2 +-
...marize.py => bins_per_sample_summarize.py} | 4 +-
...ge_abundance_and_functional_annotations.py | 20 +++-----
bin/merge_annotations.py | 2 +-
bin/merge_kaiju_results.py | 19 +++----
...clusters.py => quantification_clusters.py} | 49 +++++++------------
modules/assign_taxonomy.nf | 2 +-
modules/feature_counts.nf | 2 +-
modules/sum_up_bins_informations.nf | 4 +-
9 files changed, 41 insertions(+), 63 deletions(-)
rename bin/{aln2taxaffi.py => aln_to_tax_affi.py} (99%)
rename bin/{Bins_per_sample_summarize.py => bins_per_sample_summarize.py} (99%)
rename bin/{Quantification_clusters.py => quantification_clusters.py} (88%)
diff --git a/bin/aln2taxaffi.py b/bin/aln_to_tax_affi.py
similarity index 99%
rename from bin/aln2taxaffi.py
rename to bin/aln_to_tax_affi.py
index 241d836..6635297 100755
--- a/bin/aln2taxaffi.py
+++ b/bin/aln_to_tax_affi.py
@@ -1,7 +1,7 @@
#!/usr/bin/env python3
"""----------------------------------------------------------------------------
- Script Name: aln2taxaffi.py
+ Script Name: aln_to_tax_affi.py
Description:
Input files: File with correspondence between accession ids and taxon ids, \
taxonomy directory and diamond output file (.m8)
diff --git a/bin/Bins_per_sample_summarize.py b/bin/bins_per_sample_summarize.py
similarity index 99%
rename from bin/Bins_per_sample_summarize.py
rename to bin/bins_per_sample_summarize.py
index 89efa2b..08e7112 100755
--- a/bin/Bins_per_sample_summarize.py
+++ b/bin/bins_per_sample_summarize.py
@@ -1,7 +1,7 @@
#!/usr/bin/env python
"""----------------------------------------------------------------------------
- Script Name: Bins_per_sample.py
+ Script Name: bins_per_sample_summarize.py
Description: Generate abundances table of bins between samples, with also \
taxonomic and genomes informations from gtdb-tk and Checkm2.
Input files: Samtools coverages anf flagstats files per sample, .fasta file of bins, \
@@ -307,4 +307,4 @@ def main():
write_report_file(general_table, args.report_file, args.checkm_file, args.table_file)
if __name__ == '__main__':
- main()
\ No newline at end of file
+ main()
diff --git a/bin/merge_abundance_and_functional_annotations.py b/bin/merge_abundance_and_functional_annotations.py
index 3885286..614b123 100755
--- a/bin/merge_abundance_and_functional_annotations.py
+++ b/bin/merge_abundance_and_functional_annotations.py
@@ -28,18 +28,14 @@ __status__ = 'dev'
# Status: dev.
# Modules importation.
-try:
- import argparse
- import re
- import sys
- import pandas as pd
- from datetime import datetime
-except ImportError as error:
- print(error)
- exit(1)
-
-# Print time.
-print(str(datetime.now()))
+
+import argparse
+import re
+import sys
+import pandas as pd
+from datetime import datetime
+
+
# Manage parameters.
parser = argparse.ArgumentParser(description = 'Script which join \
diff --git a/bin/merge_annotations.py b/bin/merge_annotations.py
index afa038a..4b35037 100755
--- a/bin/merge_annotations.py
+++ b/bin/merge_annotations.py
@@ -268,4 +268,4 @@ def main():
if __name__ == '__main__':
- main()
\ No newline at end of file
+ main()
diff --git a/bin/merge_kaiju_results.py b/bin/merge_kaiju_results.py
index 9980126..25cdb1c 100755
--- a/bin/merge_kaiju_results.py
+++ b/bin/merge_kaiju_results.py
@@ -21,18 +21,13 @@ __status__ = 'dev'
# Status: dev.
# Modules importation.
-try:
- import argparse
- import re
- import sys
- import pandas as pd
- from datetime import datetime
-except ImportError as error:
- print(error)
- exit(1)
-
-# Print time.
-print(str(datetime.now()))
+
+import argparse
+import re
+import sys
+import pandas as pd
+from datetime import datetime
+
# Manage parameters.
parser = argparse.ArgumentParser(description = 'Script which join \
diff --git a/bin/Quantification_clusters.py b/bin/quantification_clusters.py
similarity index 88%
rename from bin/Quantification_clusters.py
rename to bin/quantification_clusters.py
index 49f4ad4..84c7367 100755
--- a/bin/Quantification_clusters.py
+++ b/bin/quantification_clusters.py
@@ -1,15 +1,14 @@
#!/usr/bin/env python
"""--------------------------------------------------------------------
- Script Name: Quantification_clusters.py
+ Script Name: quantification_clusters.py
Description: Create a file which join
table with global cluster id and intermediate cluster id
to table with intermediate cluster id and genes id.
Create a file which contains
sum of reads aligned
to each gene of a cluster.
- Input files:
- 1st input file: table_clstr.txt (table with cluster id
+ Input files: 1st input file: table_clstr.txt (table with cluster id
and corresponding intermediate cluster ids)
2nd input file: file containing list of file names
generated with 1st cd-hit for each sample
@@ -17,7 +16,7 @@
3rd input file: file containing list of file names
generated with featureCounts for each sample
(.featureCounts.count files)
- Created By: Joanna Fourquet et Celine Noirot
+ Created By: Joanna Fourquet and Celine Noirot
Date: 2019-04-11
-----------------------------------------------------------------------
"""
@@ -34,14 +33,11 @@ __status__ = 'dev'
# Status: dev.
# Modules importation.
-try:
- import argparse
- import re
- import sys
- from datetime import datetime
-except ImportError as error:
- print(error)
- exit(1)
+import argparse
+import re
+import sys
+from datetime import datetime
+
# Print time.
print(str(datetime.now()))
@@ -52,22 +48,22 @@ correspondence table between global cluster id and gene id and \
a table with number of aligned reads in each sample and for each \
global cluster id.')
-parser.add_argument('-t', '--table_of_corespondences', required = True, \
+parser.add_argument('-t', '--table_of_corespondences', required = True,
help = 'Correspondence table between global cluster \
id and intermediate cluster id.')
-parser.add_argument('-l', '--list_of_file_clusters', required = True, \
+parser.add_argument('-l', '--list_of_file_clusters', required = True,
help = 'List of files containing correspondence tables between \
cluster intermediate cluster id and gene id per sample.')
-parser.add_argument('-c', '--list_of_file_counts', required = True, \
+parser.add_argument('-c', '--list_of_file_counts', required = True,
help = 'List of files storing read counts for each gene per sample.')
-parser.add_argument('-oc', '--output_counts', required = True, \
+parser.add_argument('-oc', '--output_counts', required = True,
help = 'Name of output file containing counts \
for each global cluster id and each sample.')
-parser.add_argument('-oid', '--output_id', required = True, \
+parser.add_argument('-oid', '--output_id', required = True,
help = 'Name of output file containing correspondence table \
between global cluster id and gene id.')
@@ -100,8 +96,6 @@ with open(args.table_of_corespondences) as fp:
print(d_g_clstr_id_by_int_clstr_id)
print(d_count_by_g_clstr)
-# Print date.
-print(str(datetime.now()))
# Initialization of dictionnary d_g_clstr_id_by_gene_id.
d_g_clstr_id_by_gene_id = {}
@@ -127,10 +121,7 @@ for int_clstr_gene_path in files_of_int_clstr_id_gene_id:
print(line_int_clstr_gene)
int_clstr_id = line_int_clstr_gene[0]
gene_id_from_clstr_gene_path = line_int_clstr_gene[1]
- if \
- 'd_g_clstr_id_by_gene_id[gene_id_from_clstr_gene_path]' \
- not in d_g_clstr_id_by_gene_id:
- print("if")
+ if 'd_g_clstr_id_by_gene_id[gene_id_from_clstr_gene_path]' not in d_g_clstr_id_by_gene_id:
d_g_clstr_id_by_gene_id[gene_id_from_clstr_gene_path] \
= d_g_clstr_id_by_int_clstr_id[int_clstr_id]
else:
@@ -139,8 +130,7 @@ for int_clstr_gene_path in files_of_int_clstr_id_gene_id:
print(d_g_clstr_id_by_gene_id)
-# Print date.
-print(str(datetime.now()))
+
# For each count file (output of featureCounts), reading of lines one by one,
# recovery of name of gene and count number and incrementing of corresponding
@@ -157,8 +147,7 @@ for (count_idx,counts_path) in enumerate(sorted(files_of_counts)):
d_count_by_g_clstr[d_g_clstr_id_by_gene_id[gene_id]]\
[count_idx] += gene_count
-# Print date.
-print(str(datetime.now()))
+
#######################################
# Write in the output files.
@@ -178,8 +167,7 @@ with open(args.output_id,"w") as foutput_res_table:
+ gene_id \
+ "\n")
-# Print date.
-print(str(datetime.now()))
+
# Write output file containing global cluster id and read count for each sample.
with open(args.output_counts,"w") as foutput_res_counts:
@@ -192,5 +180,4 @@ with open(args.output_counts,"w") as foutput_res_counts:
+ "\t".join([str(i) for i in count])\
+ "\n")
-# Print date.
-print(str(datetime.now()))
+
diff --git a/modules/assign_taxonomy.nf b/modules/assign_taxonomy.nf
index 559c06c..9634f94 100644
--- a/modules/assign_taxonomy.nf
+++ b/modules/assign_taxonomy.nf
@@ -38,7 +38,7 @@ process ASSIGN_TAXONOMY {
fi
- aln2taxaffi.py -a ${accession2taxid} --taxonomy \$new_taxdump_var \
+ aln_to_tax_affi.py -a ${accession2taxid} --taxonomy \$new_taxdump_var \
-o ${meta.id} -b ${m8} --keep_only_best_aln \
-v --write_top_taxons
merge_contig_quantif_perlineage.py -c ${meta.id}.percontig.tsv -s ${sam_coverage} -o ${meta.id}
diff --git a/modules/feature_counts.nf b/modules/feature_counts.nf
index 97587cc..73ec38c 100644
--- a/modules/feature_counts.nf
+++ b/modules/feature_counts.nf
@@ -37,7 +37,7 @@ process QUANTIFICATION_TABLE {
"""
ls ${clusters_contigs} | cat > List_of_contigs_files.txt
ls ${counts_files} | cat > List_of_count_files.txt
- Quantification_clusters.py -t ${global_clusters_clusters} -l List_of_contigs_files.txt -c List_of_count_files.txt -oc Clusters_Count_table_all_samples.txt -oid Correspondence_global_clstr_genes.txt
+ quantification_clusters.py -t ${global_clusters_clusters} -l List_of_contigs_files.txt -c List_of_count_files.txt -oc Clusters_Count_table_all_samples.txt -oid Correspondence_global_clstr_genes.txt
"""
}
diff --git a/modules/sum_up_bins_informations.nf b/modules/sum_up_bins_informations.nf
index d09a2a1..842b43c 100644
--- a/modules/sum_up_bins_informations.nf
+++ b/modules/sum_up_bins_informations.nf
@@ -18,7 +18,7 @@ process GENOMES_ABUNDANCES_PER_SAMPLE {
script:
"""
mkdir -p stats
- Bins_per_sample_summarize.py --list_of_coverage_files ${coverage_files} \
+ bins_per_sample_summarize.py --list_of_coverage_files ${coverage_files} \
--list_of_flagstats_files ${flagstats_files} --affiliations_predictions ${affiliations_predictions} \
--bins_folder ${bins_folder} --genomes_informations ${genomes_informations} \
--output_file genomes_abundances.tsv --report_file stats/genomes_abundances_mqc.tsv \
@@ -76,4 +76,4 @@ process BINS_STATS_TO_MUTLIQC_FORMAT {
cat bin_size_per_quality.tsv >> bin_size_per_quality_mqc.tsv
"""
-}
\ No newline at end of file
+}
--
GitLab
From 33eb4300f86ba892fd23658857a3f16a38a011f0 Mon Sep 17 00:00:00 2001
From: VIENNE MAINA <maina.vienne@inrae.fr>
Date: Tue, 8 Nov 2022 10:53:03 +0100
Subject: [PATCH 146/194] Fix filter_diamond_hits.py
---
bin/filter_diamond_hits.py | 5 ++---
1 file changed, 2 insertions(+), 3 deletions(-)
diff --git a/bin/filter_diamond_hits.py b/bin/filter_diamond_hits.py
index 375542a..a29e514 100755
--- a/bin/filter_diamond_hits.py
+++ b/bin/filter_diamond_hits.py
@@ -68,9 +68,8 @@ def get_all_hits_per_query(blast_result_file):
def is_identity_and_coverage_ok(hit, min_identity, min_coverage):
qcovhsp = (int(hit["qend"]) - int(hit["qstart"]) + 1) / int(hit['qlen'])
- if float(hit['pident']) >= min_identity or qcovhsp >= min_coverage:
- return True
- return False
+ return float(hit['pident']) >= min_identity and qcovhsp >= min_coverage
+
def parse_arguments():
--
GitLab
From c1348c83c82fa721496030787c11f06e542f7a58 Mon Sep 17 00:00:00 2001
From: Vincent Darbot <vincent.darbot@inrae.fr>
Date: Tue, 8 Nov 2022 11:25:35 +0100
Subject: [PATCH 147/194] pep8 modifs
---
bin/aln_to_tax_affi.py | 56 ++++++++++----------
bin/bins_per_sample_summarize.py | 90 +++++++++++++++++---------------
2 files changed, 78 insertions(+), 68 deletions(-)
diff --git a/bin/aln_to_tax_affi.py b/bin/aln_to_tax_affi.py
index 6635297..a887609 100755
--- a/bin/aln_to_tax_affi.py
+++ b/bin/aln_to_tax_affi.py
@@ -82,15 +82,15 @@ def parse_arguments():
help="Keep only diamond alignments with top alignment score. (overrides --top)", action="store_true")
parser.add_argument('--write_top_taxons',
- help="""Write top taxons per contig for each rank
+ help="""Write top taxons per contig for each rank
with their weigth associated in 'top_taxons_per_contig.tsv'.
- Can be helpful to understand the affiliations made.""",
+ Can be helpful to understand the affiliations made.""",
action="store_true")
-
+
parser.add_argument('--write_top_taxons_verbose',
- help="""Write top taxons per contig for each rank
+ help="""Write top taxons per contig for each rank
with their weigth associated in a verbose mode 'top_taxons_per_contig_verbose.tsv'.
- Can be helpful to understand the affiliations made.""",
+ Can be helpful to understand the affiliations made.""",
action="store_true")
parser.add_argument("--query_length_file",
@@ -357,14 +357,15 @@ def get_taxid_consensus(collate_table, main_ranks):
if dP > MIN_FRACTION:
logging.debug(f'-->dP OK {best_taxid}')
return best_taxid
- #(fullnamelineage_text, fullnamelineage_ids) = d_taxonomy[str(sortCollate[0][0])].lineage_main_level()
- #tax_id_keep = str(sortCollate[0][0])
+ # (fullnamelineage_text, fullnamelineage_ids) = d_taxonomy[str(sortCollate[0][0])].lineage_main_level()
+ # tax_id_keep = str(sortCollate[0][0])
# return (tax_id_keep, fullnamelineage_text, fullnamelineage_ids)
return 1 # (1," Unable to find taxonomy consensus",1)
+
def get_top_taxid(collate_table, main_ranks):
top_taxons_per_rank = {}
-
+
for rank in main_ranks[::-1]:
collate = collate_table[rank]
if not collate:
@@ -374,9 +375,9 @@ def get_top_taxid(collate_table, main_ranks):
sortCollate = sorted(list(collate.items()), key=operator.itemgetter(1), reverse=True)
logging.debug(f"{rank}, {sortCollate}, sum score {dWeight}")
- top_taxids = [(taxid, taxid_score/dWeight) for taxid, taxid_score in sortCollate if taxid_score/dWeight > 0.01]
+ top_taxids = [(taxid, taxid_score/dWeight) for taxid, taxid_score in sortCollate if taxid_score/dWeight > 0.01]
top_taxons_per_rank[rank] = top_taxids
-
+
return top_taxons_per_rank # (1," Unable to find taxonomy consensus",1)
@@ -403,7 +404,7 @@ def get_affilaition_line(contig, taxid, taxid2rankedlineage, taxid2name):
def plot_taxonomic_assignment(output_name, count_genealogy, count_genealogy_contig, nb_total_prot, nb_prot_annotated, nb_prot_assigned):
- # graphs
+ # graphs
try:
os.makedirs("graphs")
except OSError:
@@ -442,32 +443,34 @@ def plot_taxonomic_assignment(output_name, count_genealogy, count_genealogy_con
def get_top_taxons_info(contig, top_taxons_per_rank, taxid2name):
- info = {'contig':contig,}
+ info = {'contig': contig, }
for rank, top_taxids in top_taxons_per_rank.items():
- top_affi_by_rank = []
+ top_affi_by_rank = []
for taxid, score in top_taxids:
taxname_and_weigth = f"{taxid2name[taxid]} ({100*score:.1f})"
top_affi_by_rank.append(taxname_and_weigth)
- info[rank] = ';'.join(top_affi_by_rank)
+ info[rank] = ';'.join(top_affi_by_rank)
return info
-
+
+
def get_top_taxons_info_verbose(contig, top_taxons_per_rank, taxid2name, taxid2rankedlineage):
list_info = []
for rank, top_taxids in top_taxons_per_rank.items():
-
+
for taxid, score in top_taxids:
-
+
lienage = [taxid2name[tid] for tid in taxid2rankedlineage[taxid] if tid!="None"]
- info= {"contig":contig,
- "rank":rank,
- "lineage": ';'.join(lienage),
- 'taxon':taxid2name[taxid],
- "score":round(100 * score,1)
- }
+ info= {"contig": contig,
+ "rank": rank,
+ "lineage": ';'.join(lienage),
+ 'taxon': taxid2name[taxid],
+ "score": round(100 * score, 1)
+ }
list_info.append(info)
return list_info
+
def main():
args = parse_arguments()
@@ -529,7 +532,7 @@ def main():
contig2matches = group_by_contig(matches, re_contig)
top_taxon_infos = []
top_taxon_infos_verbose = []
-
+
with open(output_name + ".pergene.tsv", "w") as out_protein, \
open(output_name + ".percontig.tsv", "w") as out_contig, \
open(output_name + ".warn.tsv", "w") as outdisc:
@@ -579,9 +582,9 @@ def main():
outdisc.write(f"{protein_id}\tNo protid correspondance file\t{','.join(sorted(accessions_unfound_in_mapping_prot))}\n")
consensual_contig_taxid = get_taxid_consensus(contig_collate_hits, main_ranks)
- if args.write_top_taxons or args.write_top_taxons_verbose:
+ if args.write_top_taxons or args.write_top_taxons_verbose:
top_taxons_per_rank = get_top_taxid(contig_collate_hits, main_ranks)
-
+
if args.write_top_taxons:
top_taxon_info = get_top_taxons_info(contig, top_taxons_per_rank, taxid2name)
top_taxon_infos.append(top_taxon_info)
@@ -596,7 +599,6 @@ def main():
out_contig.write(contig_affi_line)
logging.debug(contig_affi_line)
-
if query_length_file:
logging.debug("Plot taxonomic affiliation using protein lengths.")
with open(query_length_file) as fl:
diff --git a/bin/bins_per_sample_summarize.py b/bin/bins_per_sample_summarize.py
index 08e7112..13493b8 100755
--- a/bin/bins_per_sample_summarize.py
+++ b/bin/bins_per_sample_summarize.py
@@ -36,6 +36,7 @@ import os
# Function
################################################
+
def bins_contigs_compositions(folder):
'''
- Function: Associate each contig with the bin were the contig is retrieved.
@@ -57,6 +58,7 @@ def bins_contigs_compositions(folder):
return contigs_to_bins, list_bins
+
def calculate_sample_bins_abundances(list_of_coverage_files, list_of_flagstat_files, contigs_to_bins, list_bins):
'''
- Function: Create a panda datadrame of bins abundances with sample as columns, and bins as rows.
@@ -72,38 +74,38 @@ def calculate_sample_bins_abundances(list_of_coverage_files, list_of_flagstat_fi
'''
list_samples = list()
for coverage_file in list_of_coverage_files:
- sample_name = coverage_file.replace('.coverage.tsv','')
+ sample_name = coverage_file.replace('.coverage.tsv', '')
list_samples.append(sample_name)
-
+
list_of_df_by_bin = list()
for coverage_file in list_of_coverage_files:
- sample_name = coverage_file.replace('.coverage.tsv','')
+ sample_name = coverage_file.replace('.coverage.tsv', '')
df = pd.read_csv(coverage_file, sep='\t')
df['bin'] = df['#rname'].apply(lambda c: contigs_to_bins[c])
df['total_depth'] = df['meandepth'] * df['endpos']
- df_by_bin = df.groupby(['bin']).agg({"#rname":'count', #"#rname":';'.join,
- 'numreads':sum,
- 'endpos':sum,
- "total_depth":sum,
+ df_by_bin = df.groupby(['bin']).agg({"#rname": 'count', #"#rname":';'.join,
+ 'numreads': sum,
+ 'endpos': sum,
+ "total_depth": sum,
}).reset_index()
- df_by_bin = df_by_bin.rename(columns={"#rname": "contig_count", "endpos":'bin_size'})
+ df_by_bin = df_by_bin.rename(columns={"#rname": "contig_count", "endpos": 'bin_size'})
df_by_bin = df_by_bin.set_index('bin')
df_by_bin['meandepth'] = df_by_bin["total_depth"]/df_by_bin["bin_size"]
df_by_bin = df_by_bin.drop(["total_depth"], axis=1)
- df_by_bin = df_by_bin.rename(columns={"numreads" : f"numreads_{sample_name}", "meandepth" : f"meandepth_{sample_name}"})
+ df_by_bin = df_by_bin.rename(columns={"numreads": f"numreads_{sample_name}", "meandepth": f"meandepth_{sample_name}"})
list_of_df_by_bin.append(df_by_bin)
sample_to_bins_abundances = pd.concat(list_of_df_by_bin, axis=1)
- # Add number of unassigned reads in the table
+ # Add number of unassigned reads in the table
flagstat_regexes = {
- "primary": r"(\d+) \+ (\d+) primary",
- "primary_mapped": r"(\d+) \+ (\d+) primary mapped \((.+):(.+)\)"
- }
+ "primary": r"(\d+) \+ (\d+) primary",
+ "primary_mapped": r"(\d+) \+ (\d+) primary mapped \((.+):(.+)\)"
+ }
for flagstat_file in list_of_flagstat_files:
- sample_name = flagstat_file.replace('.flagstat','')
+ sample_name = flagstat_file.replace('.flagstat', '')
total = 0
mapped = 0
with open(flagstat_file, 'r') as fi:
@@ -115,25 +117,26 @@ def calculate_sample_bins_abundances(list_of_coverage_files, list_of_flagstat_fi
elif r_search and flag == "primary_mapped":
mapped = int(r_search.group(0).split()[0])
- sample_to_bins_abundances.loc['unmapped_to_bin',f"numreads_{sample_name}"] = \
+ sample_to_bins_abundances.loc['unmapped_to_bin', f"numreads_{sample_name}"] = \
total - mapped
- columns_numreads = [ c for c in sample_to_bins_abundances.columns if c.startswith('numreads')]
- columns_meandepth = [ c for c in sample_to_bins_abundances.columns if c.startswith('meandepth')]
+ columns_numreads = [c for c in sample_to_bins_abundances.columns if c.startswith('numreads')]
+ columns_meandepth = [c for c in sample_to_bins_abundances.columns if c.startswith('meandepth')]
sample_to_bins_abundances['sum_numreads'] = sample_to_bins_abundances[columns_numreads].sum(axis=1)
sample_to_bins_abundances['sum_meandepth'] = sample_to_bins_abundances[columns_meandepth].sum(axis=1)
sample_to_bins_abundances = sample_to_bins_abundances.drop(['bin_size'], axis=1)
- sample_to_bins_abundances = sample_to_bins_abundances.loc[:,~sample_to_bins_abundances.columns.duplicated()]
+ sample_to_bins_abundances = sample_to_bins_abundances.loc[:, ~sample_to_bins_abundances.columns.duplicated()]
return sample_to_bins_abundances
+
def add_genomes_informations(genome_info, final_bins):
'''
- Function: Adds bins informations metrics (completeness,contamination,length,N50)
- Input: drep/data_tables/genomeInformation.csv file generated previously.
'''
df_drep = pd.read_csv(genome_info)
- df_drep = df_drep.rename(columns = {'N50':'genome_N50', 'length':'genome_length'})
+ df_drep = df_drep.rename(columns = {'N50': 'genome_N50', 'length': 'genome_length'})
df_drep['genome'] = df_drep['genome'].str.split('.').str[:-1].str.join('.')
df_drep = df_drep.loc[df_drep['genome'].isin(final_bins)]
df_drep = df_drep.drop(['centrality'], axis=1)
@@ -141,6 +144,7 @@ def add_genomes_informations(genome_info, final_bins):
return df_drep
+
def return_lowest_taxo_rank(taxo):
'''
- Function: Returns the lowest non-null taxonomic affiliation from the entire taxonomic \
@@ -148,27 +152,29 @@ def return_lowest_taxo_rank(taxo):
- Input: d__Bacteria;p__Firmicutes_A;c__Clostridia;o__Clostridiales;f__Clostridiaceae;g__Clostridium;s__Clostridium beijerinckii
- Output: s__Clostridium beijerinckii
'''
- ranks = ["s__","g__","f__","o__","c__","p__","d__"]
+ ranks = ["s__", "g__", "f__", "o__", "c__", "p__", "d__"]
if not type(taxo) == float:
for i in range(-1, -len(ranks)-1, -1):
if taxo.split(';')[i] != ranks[-i-1]:
return taxo.split(';')[i]
return "unknown"
+
def add_bins_affiliations(affiliations_predictions_file):
'''
- Function: Reads gtdb-tk taxonomic affiliations file and add sample_name associated.
Splits the classificiations column into differents rank taxonomic columns.
'''
- affiliations = pd.read_csv(affiliations_predictions_file, sep='\t', usecols = ['user_genome','classification']).set_index('user_genome')
+ affiliations = pd.read_csv(affiliations_predictions_file, sep='\t', usecols = ['user_genome', 'classification']).set_index('user_genome')
affiliations['genome_name'] = affiliations['classification'].apply(lambda taxo: return_lowest_taxo_rank(taxo))
- ranks = { 0: "Domain", 1: "Phylum", 2: "Class", 3: "Order", 4: "Family", 5: "Genus", 6: "Species"}
- for i,cur_rank in ranks.items():
+ ranks = {0: "Domain", 1: "Phylum", 2: "Class", 3: "Order", 4: "Family", 5: "Genus", 6: "Species"}
+ for i, cur_rank in ranks.items():
affiliations[cur_rank] = affiliations['classification'].str.split(';').str[i].str.split('__').str[-1]
- affiliations = affiliations[ ['genome_name'] + ['Domain'] + ["Phylum"] + ["Class"] + ["Order"] + ["Family"] + ["Genus"] + ["Species"] ]
+ affiliations = affiliations[['genome_name'] + ['Domain'] + ["Phylum"] + ["Class"] + ["Order"] + ["Family"] + ["Genus"] + ["Species"]]
return affiliations
+
def write_general_output_file(affiliations, informations, abundances, output_file):
'''
- Function: Concatanate abundances, genomes informations and affiliations DataFrames \
@@ -177,15 +183,16 @@ def write_general_output_file(affiliations, informations, abundances, output_fil
'''
bins_general = pd.concat([affiliations, informations, abundances], axis=1)
bins_general = \
- bins_general.sort_values('sum_numreads', ascending=False )
+ bins_general.sort_values('sum_numreads', ascending=False)
bins_general['genome_id'] = bins_general.index
bins_general = bins_general[['genome_id'] + [c for c in bins_general.columns if c != "genome_id"]]
bins_general.loc[bins_general['genome_id'] == "unmapped_to_bin", 'genome_name'] = 'unmapped_to_bin'
- bins_general.to_csv(output_file,sep='\t', index=False)
+ bins_general.to_csv(output_file, sep='\t', index=False)
return bins_general
-def write_report_file(general_table , report_file, checkm2_file, table_file):
+
+def write_report_file(general_table, report_file, checkm2_file, table_file):
'''
- Function: Write mqc files in order to make MultiQC output figures.
'''
@@ -195,12 +202,12 @@ def write_report_file(general_table , report_file, checkm2_file, table_file):
idx.remove('unmapped_to_bin')
report_df = report_df.reindex(idx + ['unmapped_to_bin'])
report_df = report_df.set_index('genome_name')
- report_cols = [col for col in report_df.columns if col.startswith('numreads') or col == "sum_numreads"]
- table_cols = [col for col in table_df.columns if not col.startswith('meandepth_') and not col.startswith('numreads_') \
+ report_cols = [col for col in report_df.columns if col.startswith('numreads') or col == "sum_numreads"]
+ table_cols = [col for col in table_df.columns if not col.startswith('meandepth_') and not col.startswith('numreads_') \
and not col == "Domain" and not col == "Phylum" and not col == "Class" and not col == "Order" \
- and not col == "Family" and not col == "Genus" and not col == "Species"]
+ and not col == "Family" and not col == "Genus" and not col == "Species"]
table_df = table_df[table_cols]
- table_df = table_df[(table_df['completeness'] > 50 ) & (table_df['contamination'] < 10)]
+ table_df = table_df[(table_df['completeness'] > 50) & (table_df['contamination'] < 10)]
report_df = report_df[report_cols]
# Normalize library size by transforming values as samples percentages abundances
for column in report_df.columns:
@@ -212,7 +219,7 @@ def write_report_file(general_table , report_file, checkm2_file, table_file):
# filter = ((report_df>=5).any()) | (report_df.columns == "unmapped_to_bin")
unmapped_col = report_df["unmapped_to_bin"]
report_df = report_df.iloc[:, :30]
- report_df.loc[:,"unmapped_to_bin"] = unmapped_col
+ report_df.loc[:, "unmapped_to_bin"] = unmapped_col
##
report_df.index.name = "sample"
report_df = report_df.reset_index(level='sample')
@@ -220,21 +227,21 @@ def write_report_file(general_table , report_file, checkm2_file, table_file):
table_df.to_csv(table_file, sep="\t", index=False)
### generate .json file for checkm2 quality bins scatterplot
checkm_to_json = dict(id = 'bins_quality',
- section_name = 'Bins Quality overview',
- description = "Quality of bins in terms of completeness and contamination calculated by Checkm2. The points are colored according to their quality, according to the MIMAG standards defined previously (see Bins Counts quality section). Genomes with the best quality (100\% completeness and 0\% contamination) are located in the lower right corner of the graph. ",
- plot_type = 'scatter',
- anchor = 'bins_quality',
- pconfig = dict(
- title = 'Bins quality overview',
- ylab = 'Contamination',
- xlab = 'Completeness'))
+ section_name = 'Bins Quality overview',
+ description = "Quality of bins in terms of completeness and contamination calculated by Checkm2. The points are colored according to their quality, according to the MIMAG standards defined previously (see Bins Counts quality section). Genomes with the best quality (100\% completeness and 0\% contamination) are located in the lower right corner of the graph. ",
+ plot_type = 'scatter',
+ anchor = 'bins_quality',
+ pconfig = dict(
+ title = 'Bins quality overview',
+ ylab = 'Contamination',
+ xlab = 'Completeness'))
conditions = [
((general_table['completeness'] > 90) & (general_table['contamination'] < 5)),
((general_table['completeness'] > 50) & (general_table['contamination'] < 10)),
((general_table['completeness'] < 50) | (general_table['contamination'] > 10))
]
# create a list of color according to the quality of the bins
- values = ['#D5ECC2' , '#FFD3B4', '#ffd92f']
+ values = ['#D5ECC2', '#FFD3B4', '#ffd92f']
general_table['color'] = np.select(conditions, values)
checkm_to_json['data'] = dict()
@@ -246,6 +253,7 @@ def write_report_file(general_table , report_file, checkm2_file, table_file):
f.write(json_data)
###
+
def parse_arguments():
# Manage parameters.
parser = argparse.ArgumentParser()
--
GitLab
From 29fe91c89b335583a78a61ed8d78b3d12685b81c Mon Sep 17 00:00:00 2001
From: Vincent Darbot <vincent.darbot@inrae.fr>
Date: Tue, 8 Nov 2022 11:56:18 +0100
Subject: [PATCH 148/194] autopep8
---
...ge_abundance_and_functional_annotations.py | 56 +++----
bin/merge_annotations.py | 141 ++++++++++--------
2 files changed, 109 insertions(+), 88 deletions(-)
mode change 100755 => 100644 bin/merge_abundance_and_functional_annotations.py
mode change 100755 => 100644 bin/merge_annotations.py
diff --git a/bin/merge_abundance_and_functional_annotations.py b/bin/merge_abundance_and_functional_annotations.py
old mode 100755
new mode 100644
index 614b123..8cb1365
--- a/bin/merge_abundance_and_functional_annotations.py
+++ b/bin/merge_abundance_and_functional_annotations.py
@@ -30,35 +30,31 @@ __status__ = 'dev'
# Modules importation.
import argparse
-import re
-import sys
import pandas as pd
-from datetime import datetime
-
# Manage parameters.
-parser = argparse.ArgumentParser(description = 'Script which join \
+parser = argparse.ArgumentParser(description='Script which join \
quantification table by gene and tables by samples \
with functional annotations')
-parser.add_argument('-t', '--table_of_abundances', required = True, \
-help = 'Table containing counts \
+parser.add_argument('-t', '--table_of_abundances', required=True,
+ help='Table containing counts \
for each global gene id in each sample.')
-parser.add_argument('-f', '--list_of_file_annotations', required = True, \
-help = 'List of files storing functional annotation for each gene per sample.')
+parser.add_argument('-f', '--list_of_file_annotations', required=True,
+ help='List of files storing functional annotation for each gene per sample.')
-parser.add_argument('-d', '--list_of_file_diamond', required = True, \
-help = 'List of files storing diamond results with best bitscore \
+parser.add_argument('-d', '--list_of_file_diamond', required=True,
+ help='List of files storing diamond results with best bitscore \
for each gene per sample.')
-parser.add_argument('-o', '--output_file', required = True, \
-help = 'Name of output file containing counts \
+parser.add_argument('-o', '--output_file', required=True,
+ help='Name of output file containing counts \
for each global gene id and its functional annotation.')
-parser.add_argument('-v', '--version', action = 'version', \
-version = __version__)
+parser.add_argument('-v', '--version', action='version',
+ version=__version__)
args = parser.parse_args()
@@ -76,28 +72,36 @@ with open(args.list_of_file_diamond) as fdiamond_list:
concat_eggnog_mapper_files = pd.DataFrame()
# Concatenate annotation files.
-for (annotations_idx,annotations_path) in enumerate(sorted(files_of_annotations)):
- eggnog_mapper_file = pd.read_csv(annotations_path, delimiter='\t', decimal='.',skiprows=4)
- concat_eggnog_mapper_files = pd.concat([concat_eggnog_mapper_files, eggnog_mapper_file])
+for (annotations_idx, annotations_path) in enumerate(sorted(files_of_annotations)):
+ eggnog_mapper_file = pd.read_csv(
+ annotations_path, delimiter='\t', decimal='.', skiprows=4)
+ concat_eggnog_mapper_files = pd.concat(
+ [concat_eggnog_mapper_files, eggnog_mapper_file])
# Creates a new empty dataframe for diamond results.
concat_diamond_files = pd.DataFrame()
# Concatenate diamond files.
-for (diamond_idx,diamond_path) in enumerate(sorted(diamond_files)):
- diamond_columns = ["qseqid","sseqid","pident","length","mismatch","gapopen","qstart","qend","sstart","send","evalue","bitscore","qlen","slen","stitle"]
- diamond_file = pd.read_csv(diamond_path, delimiter='\t', decimal='.', header=None, names=diamond_columns)
- diamond_file.loc[:,"sseqid"] = 'https://www.ncbi.nlm.nih.gov/protein/' + diamond_file.loc[:,"sseqid"]
+for (diamond_idx, diamond_path) in enumerate(sorted(diamond_files)):
+ diamond_columns = ["qseqid", "sseqid", "pident", "length", "mismatch", "gapopen",
+ "qstart", "qend", "sstart", "send", "evalue", "bitscore", "qlen", "slen", "stitle"]
+ diamond_file = pd.read_csv(
+ diamond_path, delimiter='\t', decimal='.', header=None, names=diamond_columns)
+ diamond_file.loc[:, "sseqid"] = 'https://www.ncbi.nlm.nih.gov/protein/' + \
+ diamond_file.loc[:, "sseqid"]
group_diamond_file = diamond_file.groupby("qseqid")\
- .agg({"stitle" : ';'.join, "sseqid" : ','.join})\
+ .agg({"stitle": ';'.join, "sseqid": ','.join})\
.reset_index()\
.reindex(columns=diamond_file.columns)
- res_diamond_file = group_diamond_file.loc[:,["qseqid","sseqid","stitle"]]
+ res_diamond_file = group_diamond_file.loc[:, [
+ "qseqid", "sseqid", "stitle"]]
concat_diamond_files = pd.concat([concat_diamond_files, res_diamond_file])
# Merge counts, annotation and diamond results.
-merge_annot = pd.merge(counts_file,concat_eggnog_mapper_files,left_on="seed_cluster",right_on='#query', how='left')
-merge = pd.merge(merge_annot,concat_diamond_files,left_on="seed_cluster",right_on="qseqid", how='left')
+merge_annot = pd.merge(counts_file, concat_eggnog_mapper_files,
+ left_on="seed_cluster", right_on='#query', how='left')
+merge = pd.merge(merge_annot, concat_diamond_files,
+ left_on="seed_cluster", right_on="qseqid", how='left')
merge.drop('#query', inplace=True, axis=1)
merge.drop("qseqid", inplace=True, axis=1)
merge_no_nan = merge.fillna("-")
diff --git a/bin/merge_annotations.py b/bin/merge_annotations.py
old mode 100755
new mode 100644
index 4b35037..633f858
--- a/bin/merge_annotations.py
+++ b/bin/merge_annotations.py
@@ -33,25 +33,32 @@ def parse_arguments():
parser = ArgumentParser(description="...",
formatter_class=ArgumentDefaultsHelpFormatter)
- parser.add_argument('-c', '--cds', help='gff file with CDS annotations.', required=True)
+ parser.add_argument(
+ '-c', '--cds', help='gff file with CDS annotations.', required=True)
- parser.add_argument('-t', '--trna', help='gff file with tRNA annotations.', required=True)
+ parser.add_argument(
+ '-t', '--trna', help='gff file with tRNA annotations.', required=True)
- parser.add_argument('-r', '--rrna', help='gff file with rRNA annotations.', required=True)
-
- parser.add_argument('--contig_seq', help='fasta file of contigs. Needed to extract gene sequence', required=True)
+ parser.add_argument(
+ '-r', '--rrna', help='gff file with rRNA annotations.', required=True)
- parser.add_argument('--faa_file', help='fasta file of protein sequences generated by prodigal.', required=True)
+ parser.add_argument(
+ '--contig_seq', help='fasta file of contigs. Needed to extract gene sequence', required=True)
- parser.add_argument('--gff_output', help='final gff file with all annotations.', default="all_annotation.gff")
+ parser.add_argument(
+ '--faa_file', help='fasta file of protein sequences generated by prodigal.', required=True)
- parser.add_argument('--ffn_output', help='final ffn file with all annotations.', default="all_annotation.ffn")
+ parser.add_argument(
+ '--gff_output', help='final gff file with all annotations.', default="all_annotation.gff")
- parser.add_argument('--faa_output', help='final faa file with all CDS annotations in amino acid.', default="all_annotation.faa")
-
- parser.add_argument('--report_output', help='Prokka report like to be able to show annotations in multiqc.', default="annotation_report.txt")
+ parser.add_argument(
+ '--ffn_output', help='final ffn file with all annotations.', default="all_annotation.ffn")
+ parser.add_argument(
+ '--faa_output', help='final faa file with all CDS annotations in amino acid.', default="all_annotation.faa")
+ parser.add_argument(
+ '--report_output', help='Prokka report like to be able to show annotations in multiqc.', default="annotation_report.txt")
parser.add_argument("-v", "--verbose", help="increase output verbosity",
action="store_true")
@@ -59,6 +66,7 @@ def parse_arguments():
args = parser.parse_args()
return args
+
def read_file_and_ignore_hashtag(file_path):
proper_open = gzip.open if file_path.endswith('.gz') else open
@@ -68,39 +76,43 @@ def read_file_and_ignore_hashtag(file_path):
continue
yield line
+
def group_annotations_by_contigs(*gff_annotations):
- contig2annotations = defaultdict(list)
+ contig2annotations = defaultdict(list)
for annotations in gff_annotations:
for annotation in annotations:
contig2annotations[annotation['seqname']].append(annotation)
return contig2annotations
-
+
def parse_gff_file(gff_file, feature_to_keep=False):
- gff_headers = ("seqname", "_3", "feature", "start", "end", "_2", "strand", "_1", "attribute")
+ gff_headers = ("seqname", "_3", "feature", "start",
+ "end", "_2", "strand", "_1", "attribute")
- gff_annotations = csv.DictReader(read_file_and_ignore_hashtag(gff_file),
- delimiter='\t',
- fieldnames=gff_headers)
+ gff_annotations = csv.DictReader(read_file_and_ignore_hashtag(gff_file),
+ delimiter='\t',
+ fieldnames=gff_headers)
if feature_to_keep:
logging.info(f'Keeping only {feature_to_keep} feature from {gff_file}')
- gff_annotations = (annotation for annotation in gff_annotations if annotation['feature'] == feature_to_keep)
-
+ gff_annotations = (
+ annotation for annotation in gff_annotations if annotation['feature'] == feature_to_keep)
+
return gff_annotations
+
def remove_overlapping_cds(cds_file, contig2rnas):
contig_annotations = []
current_contig = None
for cds in parse_gff_file(cds_file, 'CDS'):
-
+
reading_next_contig = cds['seqname'] != current_contig
-
+
if contig_annotations and reading_next_contig:
yield current_contig, contig_annotations
@@ -110,21 +122,23 @@ def remove_overlapping_cds(cds_file, contig2rnas):
is_overlapping = False
for rna in contig2rnas[current_contig]:
- if (int(rna['end']) < int(cds['start']) or int(rna['start']) > int(cds['end'])):
- continue
-
- else: # overlap -> remove cds
- is_overlapping = True
- overlap = f"[{max(rna['start'], cds['start'])},{min(rna['end'], cds['end'])}]"
- rna_info = f"{rna['feature']} [{rna['start']}, {rna['end']}]"
- cds_info = f"CDS [{cds['start']}, {cds['end']}]"
- logging.info(f"overlap: {cds_info} overlapping with {rna_info} at {overlap} on contig={current_contig}")
+ if (int(rna['end']) < int(cds['start']) or int(rna['start']) > int(cds['end'])):
+ continue
+
+ else: # overlap -> remove cds
+ is_overlapping = True
+ overlap = f"[{max(rna['start'], cds['start'])},{min(rna['end'], cds['end'])}]"
+ rna_info = f"{rna['feature']} [{rna['start']}, {rna['end']}]"
+ cds_info = f"CDS [{cds['start']}, {cds['end']}]"
+ logging.info(
+ f"overlap: {cds_info} overlapping with {rna_info} at {overlap} on contig={current_contig}")
if not is_overlapping:
contig_annotations.append(cds)
yield current_contig, contig_annotations
+
def merging_cds_and_rna(cds_per_contig, contig2rnas):
contig_processed = []
@@ -132,16 +146,18 @@ def merging_cds_and_rna(cds_per_contig, contig2rnas):
contig_processed.append(contig)
rna_features = contig2rnas[contig]
yield contig, rna_features + cds_features
-
+
# check that all rrna contigs have been processed if not processe them
- for contig, rnas in contig2rnas.items():
+ for contig, rnas in contig2rnas.items():
if contig not in contig_processed:
logging.info(f'{contig} has only rna annotation')
yield contig, rnas
+
def add_new_ID_tag_and_Parent(gff_attributes, new_id, parent_id):
- gff_attributes_no_id = ';'.join([attr for attr in gff_attributes.split(';') if attr.split('=')[0] not in ['ID', 'Parent']])
+ gff_attributes_no_id = ';'.join([attr for attr in gff_attributes.split(
+ ';') if attr.split('=')[0] not in ['ID', 'Parent']])
return f"ID={new_id};Parent={parent_id};{gff_attributes_no_id}"
@@ -152,8 +168,9 @@ def get_tag_value(gff_attribute, tag):
return attr.split('=')[1]
return ''
+
def writing_features_to_gff_ffn_faa(annotations_per_contig, out_gff, fna_file, out_ffn, faa_file, out_faa):
-
+
contig_fa = pyfastx.Fasta(fna_file)
protein_fa = pyfastx.Fasta(faa_file)
@@ -166,12 +183,12 @@ def writing_features_to_gff_ffn_faa(annotations_per_contig, out_gff, fna_file, o
fh_gff.write(f"##sequence-region {contig}\n")
for feature in sorted(features, key=lambda x: int(x['start'])):
- gene_count[feature['feature']] += 1
+ gene_count[feature['feature']] += 1
new_id = f"{feature['seqname']}.{feature['feature']}_{gene_count[feature['feature']]}"
parent_id = f"{new_id}_gene"
start, end = int(feature['start']), int(feature['end'])
-
+
# write faa
if feature['feature'] == "CDS":
gff_id = get_tag_value(feature['attribute'], 'ID')
@@ -180,11 +197,12 @@ def writing_features_to_gff_ffn_faa(annotations_per_contig, out_gff, fna_file, o
# seq name in faa from prodigal is <contig_name>_<cds_number>
cds_number = gff_id.split('_')[-1]
cds_prodigal_id = f"{contig}_{cds_number}"
- fh_faa.write(f">{new_id} {start}-{end}\n{protein_fa[cds_prodigal_id]}\n")
-
-
- # Write gff and ffn
- feature['attribute'] = add_new_ID_tag_and_Parent(feature['attribute'], new_id, parent_id)
+ fh_faa.write(
+ f">{new_id} {start}-{end}\n{protein_fa[cds_prodigal_id]}\n")
+
+ # Write gff and ffn
+ feature['attribute'] = add_new_ID_tag_and_Parent(
+ feature['attribute'], new_id, parent_id)
# gff line with CDS/tRNA or rRNA in feature column
feature_gff_line = '\t'.join(feature.values())
@@ -194,7 +212,7 @@ def writing_features_to_gff_ffn_faa(annotations_per_contig, out_gff, fna_file, o
feature['attribute'] = f'ID={parent_id};locus_tag={new_id}'
gene_gff_line = '\t'.join(feature.values())
- fh_gff.write(f'{gene_gff_line}\n{feature_gff_line}\n')
+ fh_gff.write(f'{gene_gff_line}\n{feature_gff_line}\n')
tag_name = get_tag_value(feature['attribute'], 'Name')
@@ -204,37 +222,34 @@ def writing_features_to_gff_ffn_faa(annotations_per_contig, out_gff, fna_file, o
# minus strand: reverse complement the sequence
feature_seq = contig_fa[contig][start-1:end].antisense
- fh_ffn.write(f">{new_id} {start}-{end} {tag_name}\n{feature_seq}\n")
+ fh_ffn.write(
+ f">{new_id} {start}-{end} {tag_name}\n{feature_seq}\n")
# building prokka like report for multiqc
report = {"organism": "NA",
- "contigs": len(contig_fa),
- "bases": contig_fa.size }
+ "contigs": len(contig_fa),
+ "bases": contig_fa.size}
report.update(gene_count)
return report
-
-
-
def main():
args = parse_arguments()
if args.verbose:
- logging.basicConfig(format="%(levelname)s: %(message)s", level=logging.DEBUG)
+ logging.basicConfig(
+ format="%(levelname)s: %(message)s", level=logging.DEBUG)
logging.info('Mode verbose ON')
else:
logging.basicConfig(format="%(levelname)s: %(message)s")
-
- trna_file = args.trna
- rrna_file = args.rrna
+ trna_file = args.trna
+ rrna_file = args.rrna
cds_file = args.cds
-
fna_file = args.contig_seq
faa_file = args.faa_file
@@ -243,7 +258,6 @@ def main():
out_faa = args.faa_output
out_report = args.report_output
-
logging.info('Parsing rRNA annoations.')
rrna_annotations = parse_gff_file(rrna_file, 'rRNA')
@@ -251,21 +265,24 @@ def main():
trna_annotations = parse_gff_file(trna_file, 'tRNA')
logging.info('Grouping RNA annoations by contig.')
- contig2rnas = group_annotations_by_contigs(trna_annotations, rrna_annotations)
-
+ contig2rnas = group_annotations_by_contigs(
+ trna_annotations, rrna_annotations)
+
logging.info('Removing CDS annoations overlapping a RNA annotations.')
- unoverlapping_cds_per_contig = remove_overlapping_cds(cds_file, contig2rnas)
+ unoverlapping_cds_per_contig = remove_overlapping_cds(
+ cds_file, contig2rnas)
logging.info('Merge CDS and RNA annotations by contig.')
- annotation_per_contigs = merging_cds_and_rna(unoverlapping_cds_per_contig, contig2rnas)
+ annotation_per_contigs = merging_cds_and_rna(
+ unoverlapping_cds_per_contig, contig2rnas)
logging.info(f'Writting CDS and RNA annotations to gff file: {out_gff}.')
- report = writing_features_to_gff_ffn_faa(annotation_per_contigs, out_gff, fna_file, out_ffn, faa_file, out_faa)
-
+ report = writing_features_to_gff_ffn_faa(
+ annotation_per_contigs, out_gff, fna_file, out_ffn, faa_file, out_faa)
+
with open(out_report, "w") as fl:
- fl.write(''.join(f"{k}: {v}\n" for k,v in report.items()))
+ fl.write(''.join(f"{k}: {v}\n" for k, v in report.items()))
-
if __name__ == '__main__':
main()
--
GitLab
From 433112f4d585218d028a34cee22af33cc565e548 Mon Sep 17 00:00:00 2001
From: VIENNE MAINA <maina.vienne@inrae.fr>
Date: Tue, 8 Nov 2022 12:04:11 +0100
Subject: [PATCH 149/194] Update bin/format_bins_stat_to_multiqc.py,
bin/merge_annotations.py, bin/plot_contigs_taxonomic_affiliation.py,
bin/plot_kaiju_stat.py, bin/quantification_by_contig_lineage.py,
bin/quantification_by_functional_annotation.py, bin/db_versions.py
---
bin/db_versions.py | 16 +++++++++-----
bin/format_bins_stat_to_multiqc.py | 4 +++-
bin/merge_annotations.py | 12 +++++-----
bin/plot_contigs_taxonomic_affiliation.py | 6 ++---
bin/plot_kaiju_stat.py | 8 +++----
bin/quantification_by_contig_lineage.py | 22 ++++++++-----------
...quantification_by_functional_annotation.py | 14 +++++-------
7 files changed, 41 insertions(+), 41 deletions(-)
diff --git a/bin/db_versions.py b/bin/db_versions.py
index 3088ea3..bf762bc 100755
--- a/bin/db_versions.py
+++ b/bin/db_versions.py
@@ -5,21 +5,25 @@ import subprocess
import os.path
import gzip
-def info_db(file):
- name=file.split()[0]
- path =os.path.realpath(file.split()[1])
- if file.split()[1]=='nodes.dmp':
+def info_db(db_info_file):
+ name=db_info_file.split()[0]
+ path =os.path.realpath(db_info_file.split()[1])
+
+ if db_info_file.split()[1]=='nodes.dmp':
path=os.path.dirname(path)
+ # get db size
process = subprocess.run(['du', '-sh',path], stdout=subprocess.PIPE)
size = process.stdout.split()[0].decode('utf-8')
+
if (name == "Host_genome"):
size = f"{size} ({get_genome_seq_count(path)} seq)"
+ # get date of last modifaction
process = subprocess.run(['stat', '-c %y', path], stdout=subprocess.PIPE)
- modif = process.stdout.split()[0].decode('utf-8')
+ modif_date = process.stdout.split()[0].decode('utf-8')
- return name,size,modif,path
+ return name,size,modif_date,path
def get_genome_seq_count(genome_path):
diff --git a/bin/format_bins_stat_to_multiqc.py b/bin/format_bins_stat_to_multiqc.py
index e94ee23..05fdeb6 100755
--- a/bin/format_bins_stat_to_multiqc.py
+++ b/bin/format_bins_stat_to_multiqc.py
@@ -22,7 +22,7 @@ import pandas as pd
def parse_arguments():
- """Parse script arguments."""
+ """Parse script arguments."""bins_per_sample_summarize.py
parser = ArgumentParser(description="...",
formatter_class=ArgumentDefaultsHelpFormatter)
@@ -65,6 +65,7 @@ def main():
# remove Not_binned category when counting bins in different quality
df_ech_count = df_ech.loc[~(df_ech['genome'] == "Not_binned")]
+
df_ech_gr = df_ech_count.groupby(['Quality']).size().reset_index(name='counts')
df_ech_gr = df_ech_gr.set_index("Quality")
@@ -91,6 +92,7 @@ def main():
logging.info(f'Writing {args.out_bins_count}')
df_count.to_csv(args.out_bins_count, sep='\t')
+
logging.info(f'Writing {args.out_bins_size}')
df_size.to_csv(args.out_bins_size, sep='\t')
diff --git a/bin/merge_annotations.py b/bin/merge_annotations.py
index 4b35037..8843414 100755
--- a/bin/merge_annotations.py
+++ b/bin/merge_annotations.py
@@ -12,7 +12,7 @@ merge_annotations.py -c prodigal.gff -r barrnap.gff -t trnascan_se.gff --contig_
# Metadata
__author__ = 'Mainguy Jean - Plateforme bioinformatique Toulouse'
-__copyright__ = 'Copyright (C) 2020 INRAE'
+__copyright__ = 'Copyright (C) 2022 INRAE'
__license__ = 'GNU General Public License'
__version__ = '0.1'
__email__ = 'support.bioinfo.genotoul@inra.fr'
@@ -25,7 +25,7 @@ import gzip
import csv
from collections import defaultdict
import pyfastx
-from Bio.Seq import Seq
+
def parse_arguments():
@@ -244,16 +244,16 @@ def main():
out_report = args.report_output
- logging.info('Parsing rRNA annoations.')
+ logging.info('Parsing rRNA annotations.')
rrna_annotations = parse_gff_file(rrna_file, 'rRNA')
- logging.info('Parsing tRNA annoations.')
+ logging.info('Parsing tRNA annotations.')
trna_annotations = parse_gff_file(trna_file, 'tRNA')
- logging.info('Grouping RNA annoations by contig.')
+ logging.info('Grouping RNA annotations by contig.')
contig2rnas = group_annotations_by_contigs(trna_annotations, rrna_annotations)
- logging.info('Removing CDS annoations overlapping a RNA annotations.')
+ logging.info('Removing CDS annotations overlapping a RNA annotations.')
unoverlapping_cds_per_contig = remove_overlapping_cds(cds_file, contig2rnas)
logging.info('Merge CDS and RNA annotations by contig.')
diff --git a/bin/plot_contigs_taxonomic_affiliation.py b/bin/plot_contigs_taxonomic_affiliation.py
index 3d51a38..a170c28 100755
--- a/bin/plot_contigs_taxonomic_affiliation.py
+++ b/bin/plot_contigs_taxonomic_affiliation.py
@@ -303,7 +303,7 @@ def parse_arguments():
parser.add_argument('--output_dir', default='plots', help="Name of the output directory")
- parser.add_argument('--top_taxon', default=10, type=int, help="Plot only the top n most abundant taxa.")
+ parser.add_argument('--nb_top_taxon', default=10, type=int, help="Plot only the top n most abundant taxa.")
parser.add_argument("-v", "--verbose", help="increase output verbosity",
action="store_true")
@@ -326,7 +326,7 @@ def main():
contig_affi_files = args.affi_taxo_quantif
- top_n_taxon = args.top_taxon
+ top_n_taxon = args.nb_top_taxon
output_dir = args.output_dir
os.makedirs(output_dir, exist_ok=True)
@@ -385,7 +385,7 @@ def main():
html_figs = []
for i, (rank, fig) in enumerate(rank2fig.items()):
- include_plotly = True if i == 0 else False
+ include_plotly = i == 0
html_fig = fig.to_html(full_html=False, include_plotlyjs=include_plotly)
html_figs.append((rank, html_fig ))
diff --git a/bin/plot_kaiju_stat.py b/bin/plot_kaiju_stat.py
index 9ea7e0b..24fcde5 100755
--- a/bin/plot_kaiju_stat.py
+++ b/bin/plot_kaiju_stat.py
@@ -2,7 +2,7 @@
"""----------------------------------------------------------------------------------------------------------------------------------------------------------
Script Name: plot_kaiju_stat.py
- Description: Generates density plot distribution of kiaju match length
+ Description: Generates density plot distribution of kaiju match length
Input files:
Verbose files generated by Kaiju for each sample.
Created By: Jean Mainguy
@@ -88,7 +88,7 @@ def main():
density_df_samples = []
max_match_length = 0
- sample2dfmatchlength = {}
+ sample2df_matchlen = {}
logging.info(f'Parsing kaiju results')
for kaiju_result in kaiju_results:
@@ -98,14 +98,14 @@ def main():
df_matchlen = parse_matchlen_from_kaiju_output(kaiju_result)
max_match_length = max((max_match_length, df_matchlen['match_length'].max()))
- sample2dfmatchlength[sample_name] = df_matchlen
+ sample2df_matchlen[sample_name] = df_matchlen
x_vals = np.linspace(0,max_match_length,max_match_length) # Specifying the limits of our data
logging.info(f'Maximum match length is {max_match_length}')
logging.info(f'Computing density for each sample')
- for sample_name, df_matchlen in sample2dfmatchlength.items():
+ for sample_name, df_matchlen in sample2df_matchlen.items():
density = gaussian_kde(df_matchlen['match_length'], weights = df_matchlen['reads'])
density.covariance_factor = lambda : smoothing_parameter #Smoothing parameter
density._compute_covariance()
diff --git a/bin/quantification_by_contig_lineage.py b/bin/quantification_by_contig_lineage.py
index 3092db3..d85fb2f 100755
--- a/bin/quantification_by_contig_lineage.py
+++ b/bin/quantification_by_contig_lineage.py
@@ -22,19 +22,15 @@ __status__ = 'dev'
# Status: dev.
# Modules importation.
-try:
- import argparse
- import re
- import sys
- import pandas as pd
- import os
- from datetime import datetime
-except ImportError as error:
- print(error)
- exit(1)
-
-# Print time.
-print(str(datetime.now()))
+
+import argparse
+import re
+import sys
+import pandas as pd
+import os
+from datetime import datetime
+
+
# Manage parameters.
parser = argparse.ArgumentParser(description = 'Script which make \
diff --git a/bin/quantification_by_functional_annotation.py b/bin/quantification_by_functional_annotation.py
index a6e0e72..e9e7515 100755
--- a/bin/quantification_by_functional_annotation.py
+++ b/bin/quantification_by_functional_annotation.py
@@ -23,14 +23,12 @@ __status__ = 'dev'
# Status: dev.
# Modules importation.
-try:
- import argparse
- import re
- import sys
- import pandas as pd
-except ImportError as error:
- print(error)
- exit(1)
+
+import argparse
+import re
+import sys
+import pandas as pd
+
# Manage parameters.
parser = argparse.ArgumentParser(description = 'Create a file with \
--
GitLab
From cf543459ccdfe3a764f5fc7b06e582a6789b50bc Mon Sep 17 00:00:00 2001
From: DARBOT Vincent <vincent.darbot@inrae.fr>
Date: Tue, 15 Nov 2022 10:01:28 +0100
Subject: [PATCH 150/194] Update docs/output.md
---
docs/output.md | 4 ++--
1 file changed, 2 insertions(+), 2 deletions(-)
diff --git a/docs/output.md b/docs/output.md
index 90cb34c..4ee8815 100644
--- a/docs/output.md
+++ b/docs/output.md
@@ -164,7 +164,7 @@ The `results/` directory contains a sub-directory for each step launched:
| `SAMPLE_NAME/maxbin2`| This directory contains bins fasta files from the individual binning tool MaxBin2(see informations [here](https://academic.oup.com/bioinformatics/article/32/4/605/1744462)). |
| `SAMPLE_NAME/metabat2`| This directory contains bins fasta files from the individual binning tool MetaBAT2 (see informations [here](https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6662567/)). |
| `SAMPLE_NAME/bin_refinement`| This directory contains bins fasta files from the aggregate binning module bin_refinement from metaWRAP tool, which takes into account the bins sets from the 3 individual binning tools to try to improve the quality of the bins (see informations [here](https://microbiomejournal.biomedcentral.com/articles/10.1186/s40168-018-0541-1)). |
-| `SAMPLE_NAME/bin_refinement/unbinned_contigs.fasta`| Contigs from the sample assembly that are not retrieved in any bin. |
+| `SAMPLE_NAME/bin_refinement/unbinned_contigs.fasta`| Contigs from the sample assembly that are not retrieved in any bin. The proportion of non-binned contigs to the global size of the sample assembly can be seen in the MultiQC report, in the section Bins Size (bp) quality. |
#### **08_binning/08_2_dereplicated_bins**
| File or directory/ | Description |
@@ -172,7 +172,7 @@ The `results/` directory contains a sub-directory for each step launched:
| dereplicated_genomes/ | The dRep software allows to compare the bins of all samples in a pair-wise manner, and to clusturize the bins that share similar DNA content in terms of Average Nucleotide Identity (ANI). The threshold of 95% ANI is used to create a set of species-level representative genomes (SRGs). Within each cluster of bins, the best bin in terms of quality (completeness,contamination,N50,strain-heterogeneity) is selected as representative genome. The directory dereplicated_genomes contains all the representative bins that will be used in order to compare abundances of SRGs between the samples. |
| data_tables/genomeInformation.csv | Calculated metrics of all the bins before de-replication, used to select the representative bin after de-replication. Completeness and contamination where calculated before with CheckM2, during bin_refinement step. dRep adds further metrics and use all four to choose the best bin. |
| data_tables/Bins_clusters_composition.tsv | Associates each representative bins cluster (1st column) to the bins list within the cluster (2nd column, bins seperates by commas). |
-| figures/ | `Primary_clustering_dendrogram.pdf`: The primary clustering dendrogram summarizes the pair-wise Mash distance between all genomes in the genome list. The first clusturing used Mash, an incredibly fast but not robust algorithm, in order to accelerate the process during the second clusturing. **`Secondary_clustering_dendrograms.pdf`**: Each primary cluster with more than one member will have a page in the Secondary clustering dendrograms file. Bins with more than 95% of ANI distance similarity (--drep_threshold default value paramater, that correspond to the threshold for separate species.) will be grouped together. `Cluster_scoring.pdf`: Each secondary cluster will have its own page in the Cluster scoring figure. These figures show the score of each genome, as well as all metrics that can go into determin, and will always be the genome with the highest score.`Clustering scatterplots.pdf` provides some information about genome alignment statistics, and `Winning genomes.pdf` provides some information about only the “best†genomes of each replicate set, as well as a couple quick overall statistics.
+| figures/ | `Primary_clustering_dendrogram.pdf`: The primary clustering dendrogram summarizes the pair-wise Mash distance between all genomes in the genome list. The first clusturing used Mash, an incredibly fast but not robust algorithm, in order to accelerate the process during the second clusturing. **`Secondary_clustering_dendrograms.pdf`**: Each primary cluster with more than one member will have a page in the Secondary clustering dendrograms file. Bins with more than **95% of ANI distance similarity** (--drep_threshold default value paramater, that correspond to the threshold for separate species.) will be grouped together. **You can try different dRep thresholds to be more or less stringent, for example if the goal of dereplication is to generate a set of genomes that are distinct when mapping short reads, 98% ANI is an appropriate threshold.** You can also try higher threshold in order to separate species that belong to low divergent clades (96, or 97%). `Cluster_scoring.pdf`: Each secondary cluster will have its own page in the Cluster scoring figure. These figures show the score of each genome, as well as all metrics that can go into determin, and will always be the genome with the highest score.`Clustering scatterplots.pdf` provides some information about genome alignment statistics, and `Winning genomes.pdf` provides some information about only the “best†genomes of each replicate set, as well as a couple quick overall statistics.
#### **08_binning/08_3_gtdbtk/**
| File | Description |
--
GitLab
From 530438be5edb641bc600b2db8ee8e2df1d1edc84 Mon Sep 17 00:00:00 2001
From: Jean Mainguy <jean.mainguy@inra.fr>
Date: Mon, 21 Nov 2022 14:10:25 +0100
Subject: [PATCH 151/194] reorganise output dir of 02_assembly
---
modules/assembly.nf | 56 +++++++++++++++++-----------------
modules/metaquast.nf | 12 ++++----
modules/reads_deduplication.nf | 5 ++-
subworkflows/02_assembly.nf | 7 +++--
4 files changed, 40 insertions(+), 40 deletions(-)
diff --git a/modules/assembly.nf b/modules/assembly.nf
index 9155270..728e8b9 100644
--- a/modules/assembly.nf
+++ b/modules/assembly.nf
@@ -1,24 +1,24 @@
process METASPADES {
tag "${meta.id}"
- publishDir "${params.outdir}/02_assembly", mode: 'copy', pattern: "metaspades/*"
+ publishDir "${params.outdir}/02_assembly/02_1_primary_assembly", mode: 'copy', pattern: "metaspades_${meta.id}/*"
label 'ASSEMBLY_SR'
input:
tuple val(meta), path(reads)
output:
- tuple val(meta), path("metaspades/${meta.id}.contigs.fa"), emit: assembly
- tuple val(meta.id), path("metaspades/${meta.id}.log"), path("metaspades/${meta.id}.params.txt"), emit: report
+ tuple val(meta), path("metaspades_${meta.id}/${meta.id}.contigs.fa"), emit: assembly
+ tuple val(meta.id), path("metaspades_${meta.id}/${meta.id}.log"), path("metaspades_${meta.id}/${meta.id}.params.txt"), emit: report
path "v_spades.txt", emit: v_metaspades
script:
(_,mem,unit) = (task.memory =~ /(\d+) ([A-Z]B)/)[0]
if ( unit =~ /GB/ ) {
"""
- metaspades.py -t ${task.cpus} -m $mem -1 ${reads[0]} -2 ${reads[1]} -o metaspades
- mv metaspades/scaffolds.fasta metaspades/${meta.id}.contigs.fa
- mv metaspades/spades.log metaspades/${meta.id}.log
- mv metaspades/params.txt metaspades/${meta.id}.params.txt
+ metaspades.py -t ${task.cpus} -m $mem -1 ${reads[0]} -2 ${reads[1]} -o metaspades_${meta.id}
+ mv metaspades_${meta.id}/scaffolds.fasta metaspades_${meta.id}/${meta.id}.contigs.fa
+ mv metaspades_${meta.id}/spades.log metaspades_${meta.id}/${meta.id}.log
+ mv metaspades_${meta.id}/params.txt metaspades_${meta.id}/${meta.id}.params.txt
spades.py --version &> v_spades.txt
"""
@@ -30,22 +30,22 @@ process METASPADES {
process MEGAHIT {
tag "${meta.id}"
- publishDir "${params.outdir}/02_assembly", mode: 'copy', pattern: "megahit/*"
+ publishDir "${params.outdir}/02_assembly/02_1_primary_assembly", mode: 'copy', pattern: "megahit_${meta.id}/*"
label 'ASSEMBLY_SR'
input:
tuple val(meta), path(reads)
output:
- tuple val(meta), path("megahit/${meta.id}.contigs.fa"), emit: assembly
- tuple val(meta.id), path("megahit/${meta.id}.log"), path("megahit/${meta.id}.params.txt"), emit: report
+ tuple val(meta), path("megahit_${meta.id}/${meta.id}.contigs.fa"), emit: assembly
+ tuple val(meta.id), path("megahit_${meta.id}/${meta.id}.log"), path("megahit_${meta.id}/${meta.id}.params.txt"), emit: report
path "v_megahit.txt", emit: v_megahit
script:
"""
- megahit -t ${task.cpus} -1 ${reads[0]} -2 ${reads[1]} -o megahit --out-prefix "${meta.id}"
- mv megahit/options.json megahit/${meta.id}.params.txt
- rm -r megahit/intermediate_contigs
+ megahit -t ${task.cpus} -1 ${reads[0]} -2 ${reads[1]} -o megahit_${meta.id} --out-prefix "${meta.id}"
+ mv megahit_${meta.id}/options.json megahit_${meta.id}/${meta.id}.params.txt
+ rm -r megahit_${meta.id}/intermediate_contigs
megahit --version &> v_megahit.txt
"""
@@ -54,27 +54,27 @@ process MEGAHIT {
process HIFIASM_META {
tag "${meta.id}"
- publishDir "${params.outdir}/02_assembly", mode: 'copy', pattern: "hifiasm-meta/${meta.id}.*"
+ publishDir "${params.outdir}/02_assembly/02_1_primary_assembly", mode: 'copy', pattern: "hifiasm_meta_${meta.id}/${meta.id}.*"
label 'ASSEMBLY_HIFI'
input:
tuple val(meta), path(reads)
output:
- tuple val(meta), path("hifiasm-meta/${meta.id}.contigs.fa"), emit: assembly
- tuple val(meta.id), path("hifiasm-meta/${meta.id}.log"), path("hifiasm-meta/${meta.id}.params.txt"), emit: report
+ tuple val(meta), path("hifiasm_meta_${meta.id}/${meta.id}.contigs.fa"), emit: assembly
+ tuple val(meta.id), path("hifiasm_meta_${meta.id}/${meta.id}.log"), path("hifiasm_meta_${meta.id}/${meta.id}.params.txt"), emit: report
path "v_hifiasm_meta.txt", emit: v_hifiasm_meta
script:
"""
- mkdir hifiasm-meta
+ mkdir hifiasm_meta_${meta.id}
- hifiasm_meta -t ${task.cpus} -o ${meta.id} $reads 2> hifiasm-meta/${meta.id}.log
+ hifiasm_meta -t ${task.cpus} -o ${meta.id} $reads 2> hifiasm_meta_${meta.id}/${meta.id}.log
# gfa to fasta format
- awk '/^S/{print ">"\$2"\\n"\$3}' ${meta.id}.p_ctg.gfa | fold > hifiasm-meta/${meta.id}.contigs.fa
+ awk '/^S/{print ">"\$2"\\n"\$3}' ${meta.id}.p_ctg.gfa | fold > hifiasm_meta_${meta.id}/${meta.id}.contigs.fa
- mv ${meta.id}.cmd hifiasm-meta/${meta.id}.params.txt
+ mv ${meta.id}.cmd hifiasm_meta_${meta.id}/${meta.id}.params.txt
echo \$(hifiasm_meta --version 2>&1) > v_hifiasm_meta.txt
"""
@@ -83,25 +83,25 @@ process HIFIASM_META {
process METAFLYE {
tag "${meta.id}"
- publishDir "${params.outdir}/02_assembly", mode: 'copy', pattern: "metaflye/${meta.id}.*"
+ publishDir "${params.outdir}/02_assembly/02_1_primary_assembly", mode: 'copy', pattern: "metaflye_${meta.id}/${meta.id}.*"
label 'ASSEMBLY_HIFI'
input:
tuple val(meta), path(reads)
output:
- tuple val(meta), path("metaflye/${meta.id}.contigs.fa"), emit: assembly
- tuple val(meta.id), path("metaflye/${meta.id}.log"), path("metaflye/${meta.id}.params.json"), emit: report
+ tuple val(meta), path("metaflye_${meta.id}/${meta.id}.contigs.fa"), emit: assembly
+ tuple val(meta.id), path("metaflye_${meta.id}/${meta.id}.log"), path("metaflye_${meta.id}/${meta.id}.params.json"), emit: report
path "v_metaflye.txt", emit: v_metaflye
script:
"""
- mkdir metaflye
+ mkdir metaflye_${meta.id}
- flye --pacbio-hifi $reads -o 'metaflye' --meta -t ${task.cpus} 2> metaflye/${meta.id}.log
+ flye --pacbio-hifi $reads -o 'metaflye' --meta -t ${task.cpus} 2> metaflye_${meta.id}/${meta.id}.log
- mv metaflye/assembly.fasta metaflye/${meta.id}.contigs.fa
- mv metaflye/params.json metaflye/${meta.id}.params.json
+ mv metaflye_${meta.id}/assembly.fasta metaflye_${meta.id}/${meta.id}.contigs.fa
+ mv metaflye_${meta.id}/params.json metaflye_${meta.id}/${meta.id}.params.json
flye --version &> v_metaflye.txt
"""
@@ -109,7 +109,7 @@ process METAFLYE {
process RENAME_CONTIGS {
tag "${meta.id}"
- publishDir "${params.outdir}/02_assembly/", mode: 'copy'
+ publishDir "${params.outdir}/02_assembly/02_1_primary_assembly", mode: 'copy'
label 'PYTHON'
input:
diff --git a/modules/metaquast.nf b/modules/metaquast.nf
index dbaa508..9414136 100644
--- a/modules/metaquast.nf
+++ b/modules/metaquast.nf
@@ -1,22 +1,22 @@
process QUAST {
tag "${meta.id}"
label 'QUAST'
- publishDir "${params.outdir}", mode: 'copy', pattern: "${outdir}/${meta.id}/*"
+ publishDir "${params.outdir}/${outdir}/", mode: 'copy', pattern: "${meta.id}/*"
input:
tuple val(meta), path(assembly)
val outdir
output:
- path "${outdir}/${meta.id}/*", emit: all
- tuple val(meta), path ("${outdir}/${meta.id}/report.tsv"), emit: report
+ path "${meta.id}/*", emit: all
+ tuple val(meta), path ("${meta.id}/report.tsv"), emit: report
path "v_quast.txt", emit: v_quast
script:
"""
- mkdir -p $outdir/${meta.id}/
- touch $outdir/${meta.id}/report.tsv
- metaquast.py --threads ${task.cpus} --rna-finding --max-ref-number 0 --min-contig 0 ${assembly} -o $outdir/${meta.id} --labels ${meta.id}
+ mkdir -p ${meta.id}/
+ touch ${meta.id}/report.tsv
+ metaquast.py --threads ${task.cpus} --rna-finding --max-ref-number 0 --min-contig 0 ${assembly} -o ${meta.id} --labels ${meta.id}
quast -v &> v_quast.txt
"""
diff --git a/modules/reads_deduplication.nf b/modules/reads_deduplication.nf
index bce077d..4ff880d 100644
--- a/modules/reads_deduplication.nf
+++ b/modules/reads_deduplication.nf
@@ -1,8 +1,7 @@
process READS_DEDUPLICATION {
tag "${meta.id}"
- publishDir "${params.outdir}/02_assembly/deduplicated_reads/", mode: 'copy', pattern: '*.fastq.gz'
- publishDir "${params.outdir}/02_assembly/${meta.id}/", mode: 'copy', pattern: '*.fastq.gz'
- publishDir "${params.outdir}/02_assembly/${meta.id}/", mode: 'copy', pattern: '*.bam*'
+ publishDir "${params.outdir}/02_assembly/02_2_deduplicated_reads/", mode: 'copy', pattern: '*.fastq.gz'
+ publishDir "${params.outdir}/02_assembly/02_3_reads_vs_primary_assembly/${meta.id}/", mode: 'copy', pattern: '*.bam*'
input:
tuple val(meta), path(assembly), path(reads)
diff --git a/subworkflows/02_assembly.nf b/subworkflows/02_assembly.nf
index fe90d44..d202dbd 100644
--- a/subworkflows/02_assembly.nf
+++ b/subworkflows/02_assembly.nf
@@ -48,7 +48,7 @@ workflow STEP_02_ASSEMBLY {
}
ch_assembly_renamed = RENAME_CONTIGS(ch_assembly)
- ASSEMBLY_QUAST( ch_assembly_renamed,"02_assembly/quast_primary")
+ ASSEMBLY_QUAST( ch_assembly_renamed,"02_assembly/02_1_primary_assembly/assembly_metric/")
ch_assembly_report = ASSEMBLY_QUAST.out.report
ch_quast_v = ASSEMBLY_QUAST.out.v_quast
@@ -67,14 +67,14 @@ workflow STEP_02_ASSEMBLY {
ch_bwa_mem_v = READS_DEDUPLICATION.out.v_bwa_mem2
} else {
- MINIMAP2(ch_contigs_and_reads,"02_assembly")
+ MINIMAP2(ch_contigs_and_reads,"02_assembly/02_3_reads_vs_primary_assembly")
ch_bam = MINIMAP2.out.bam
ch_minimap2_v = MINIMAP2.out.v_minimap2
}
- GET_ALIGNMENT_METRICS(ch_bam, "02_assembly")
+ GET_ALIGNMENT_METRICS(ch_bam, "02_assembly/02_3_reads_vs_primary_assembly")
ch_idxstats = GET_ALIGNMENT_METRICS.out.sam_idxstat
ch_flagstat = GET_ALIGNMENT_METRICS.out.sam_flagstat
@@ -91,6 +91,7 @@ workflow STEP_02_ASSEMBLY {
assembly = ch_assembly_renamed
reads = ch_reads
bam = ch_bam
+
idxstats = ch_idxstats
flagstat = ch_flagstat
coverage = ch_coverage
--
GitLab
From 5cd85427a2e4618143bd5ceea648e94e854c0e34 Mon Sep 17 00:00:00 2001
From: Jean Mainguy <jean.mainguy@inra.fr>
Date: Wed, 23 Nov 2022 11:28:00 +0100
Subject: [PATCH 152/194] reorganize output directories
---
functional_tests/functions.py | 9 +++++----
functional_tests/main.py | 2 +-
main.nf | 10 +++++-----
modules/assign_taxonomy.nf | 8 ++++----
modules/diamond.nf | 2 +-
modules/filtering_cpm.nf | 14 +++++++++++---
modules/quantif_and_taxonomic_table_contigs.nf | 2 +-
subworkflows/03_filtering.nf | 8 ++++----
.../{05_alignment.nf => 05_protein_alignment.nf} | 2 +-
subworkflows/07_taxonomic_affi.nf | 4 ++--
10 files changed, 35 insertions(+), 26 deletions(-)
rename subworkflows/{05_alignment.nf => 05_protein_alignment.nf} (92%)
diff --git a/functional_tests/functions.py b/functional_tests/functions.py
index 6737586..25e7644 100644
--- a/functional_tests/functions.py
+++ b/functional_tests/functions.py
@@ -271,14 +271,15 @@ def test_file(exp_path, obs_path, method):
process = subprocess.Popen(command, stdout = subprocess.PIPE, shell = True, executable = '/bin/bash')
diff_out, error = process.communicate()
-
+
if not error:
- if diff_out.decode('ascii') != '':
+ diff_result = diff_out.decode()
+ if diff_result != '':
test = False
- out = 'Test result: Failed\nDifferences:\n{}\n'.format(diff_out.decode('ascii'))
+ out = 'Test result: Failed\nDifferences:\n{}\n'.format(diff_result)
false_cnt += 1
- elif diff_out.decode('ascii') == '':
+ elif diff_result == '':
test = True
out = 'Test result: Passed\n'
true_cnt += 1
diff --git a/functional_tests/main.py b/functional_tests/main.py
index fc8f4fb..db25d9b 100755
--- a/functional_tests/main.py
+++ b/functional_tests/main.py
@@ -42,7 +42,7 @@ steps_list = OrderedDict([
("02_assembly", 2),
("03_filtering", 3),
("04_structural_annot", 4),
- ("05_alignment", 5),
+ ("05_protein_alignment", 5),
("06_func_annot", 6),
("07_taxo_affi", 7),
("08_binning", 8)
diff --git a/main.nf b/main.nf
index 5d5d849..d9bc02b 100644
--- a/main.nf
+++ b/main.nf
@@ -16,7 +16,7 @@ include { STEP_01_CLEAN_QC as S01_CLEAN_QC } from './subworkflows/01_clean_qc'
include { STEP_02_ASSEMBLY as S02_ASSEMBLY } from './subworkflows/02_assembly'
include { STEP_03_ASSEMBLY_FILTER as S03_FILTERING } from './subworkflows/03_filtering'
include { STEP_04_STRUCTURAL_ANNOT as S04_STRUCTURAL_ANNOT } from './subworkflows/04_structural_annot'
-include { STEP_05_ALIGNMENT as S05_ALIGNMENT } from './subworkflows/05_alignment'
+include { STEP_05_PROTEIN_ALIGNMENT as S05_PROTEIN_ALIGNMENT } from './subworkflows/05_protein_alignment'
include { STEP_06_FUNC_ANNOT as S06_FUNC_ANNOT } from './subworkflows/06_functionnal_annot'
include { STEP_07_TAXO_AFFI as S07_TAXO_AFFI } from './subworkflows/07_taxonomic_affi'
include { STEP_08_BINNING as S08_BINNING } from './subworkflows/08_binning'
@@ -74,7 +74,7 @@ include { MERGE_FASTQ } from './modules/merge_fastq.nf'
S04_STRUCTURAL_ANNOT options:
--stop_at_structural_annot Stop the pipeline at this step
- S05_ALIGNMENT options:
+ S05_PROTEIN_ALIGNMENT options:
--diamond_bank Path to diamond bank used to align protein sequence of genes: "PATH/bank.dmnd".
This bank must be previously built with diamond makedb.
@@ -461,11 +461,11 @@ workflow {
ch_diamond_result = Channel.empty()
if ( !params.stop_at_clean && !params.stop_at_assembly && !params.stop_at_filtering && !params.stop_at_structural_annot && (!params.skip_func_annot || !params.skip_taxo_affi)) {
- S05_ALIGNMENT (ch_annotation_faa, ch_diamon_db)
+ S05_PROTEIN_ALIGNMENT (ch_annotation_faa, ch_diamon_db)
- ch_diamond_result = S05_ALIGNMENT.out.diamond_result
+ ch_diamond_result = S05_PROTEIN_ALIGNMENT.out.diamond_result
- ch_software_versions_S05 = S05_ALIGNMENT.out.software_versions
+ ch_software_versions_S05 = S05_PROTEIN_ALIGNMENT.out.software_versions
}
ch_quant_report = Channel.empty()
diff --git a/modules/assign_taxonomy.nf b/modules/assign_taxonomy.nf
index 9634f94..1e15530 100644
--- a/modules/assign_taxonomy.nf
+++ b/modules/assign_taxonomy.nf
@@ -1,6 +1,6 @@
process ASSIGN_TAXONOMY {
tag "${meta.id}"
- publishDir "${params.outdir}/07_taxo_affi/${meta.id}", mode: 'copy'
+ publishDir "${params.outdir}/07_taxo_affi/07_1_affiliation_per_sample/${meta.id}", mode: 'copy'
label 'PYTHON'
input:
@@ -54,7 +54,7 @@ process ASSIGN_TAXONOMY {
process PLOT_TAXONOMIC_AFFILIATIONS{
- publishDir "${params.outdir}/07_taxo_affi/", mode: 'copy'
+ publishDir "${params.outdir}/07_taxo_affi/07_3_plot/", mode: 'copy'
label 'PYTHON'
input:
@@ -62,11 +62,11 @@ process PLOT_TAXONOMIC_AFFILIATIONS{
path quantif_percontig
output:
- path "plots/*html"
+ path "*html"
script:
"""
- plot_contigs_taxonomic_affiliation.py $quantif_percontig --output_dir "plots"
+ plot_contigs_taxonomic_affiliation.py $quantif_percontig --output_dir "."
"""
diff --git a/modules/diamond.nf b/modules/diamond.nf
index 64404f8..6603b05 100644
--- a/modules/diamond.nf
+++ b/modules/diamond.nf
@@ -1,5 +1,5 @@
process DIAMOND {
- publishDir "${params.outdir}/05_alignment/05_2_database_alignment/$meta.id", mode: 'copy', pattern: "*.m8"
+ publishDir "${params.outdir}/05_protein_alignment/05_1_database_alignment/$meta.id", mode: 'copy', pattern: "*.m8"
tag "${meta.id}"
input:
diff --git a/modules/filtering_cpm.nf b/modules/filtering_cpm.nf
index cdfe8e5..5dd2c90 100644
--- a/modules/filtering_cpm.nf
+++ b/modules/filtering_cpm.nf
@@ -20,7 +20,8 @@ process MERGE_ASSEMBLY_FILTER {
label 'ASSEMBLY_FILTER'
tag "${meta.id}"
- publishDir "${params.outdir}/03_filtering/", mode: 'copy'
+ publishDir "${params.outdir}/03_filtering/03_1_filtered_assembly/", mode: 'copy', pattern: "*_select_contigs*"
+ publishDir "${params.outdir}/03_filtering/03_1_filtered_assembly/discard_contigs", mode: 'copy', pattern: "*_discard_contigs*"
input:
tuple val(meta), path(select_fasta)
@@ -36,8 +37,15 @@ process MERGE_ASSEMBLY_FILTER {
echo !{select_fasta} | sed "s/ /\\n/g" | sort > select_list
echo !{discard_fasta} | sed "s/ /\\n/g" | sort > discard_list
- for i in `cat select_list` ; do cat $i >> !{meta.id}_select_contigs_cpm!{min_cpm}.fasta ; done
- for j in `cat discard_list` ; do cat $j >> !{meta.id}_discard_contigs_cpm!{min_cpm}.fasta ; done
+ for i in `cat select_list` ;
+ do
+ cat $i >> !{meta.id}_select_contigs_cpm!{min_cpm}.fasta
+ done
+
+ for j in `cat discard_list` ;
+ do
+ cat $j >> !{meta.id}_discard_contigs_cpm!{min_cpm}.fasta
+ done
rm select_list
rm discard_list
diff --git a/modules/quantif_and_taxonomic_table_contigs.nf b/modules/quantif_and_taxonomic_table_contigs.nf
index 875df7a..faf2848 100644
--- a/modules/quantif_and_taxonomic_table_contigs.nf
+++ b/modules/quantif_and_taxonomic_table_contigs.nf
@@ -1,7 +1,7 @@
taxo_list = "all superkingdom phylum class order family genus species"
process QUANTIF_AND_TAXONOMIC_TABLE_CONTIGS {
- publishDir "${params.outdir}/07_taxo_affi", mode: 'copy'
+ publishDir "${params.outdir}/07_taxo_affi/07_2_affiliation_merged", mode: 'copy'
label 'PYTHON'
input:
diff --git a/subworkflows/03_filtering.nf b/subworkflows/03_filtering.nf
index be38cd6..3e7288b 100644
--- a/subworkflows/03_filtering.nf
+++ b/subworkflows/03_filtering.nf
@@ -66,13 +66,13 @@ workflow STEP_03_ASSEMBLY_FILTER {
ch_contigs_and_discarded_reads = ch_merged_selected.join(ch_discarded_reads)
- BWA_MEM(ch_contigs_and_discarded_reads, "03_filtering")
+ BWA_MEM(ch_contigs_and_discarded_reads, "03_filtering/03_2_reads_vs_filtered_assembly")
ch_bam_with_discarded_reads = BWA_MEM.out.bam
} else {
ch_discarded_reads = EXTRACT_SINGLE_READS(ch_discarded_bam)
ch_contigs_and_discarded_reads = ch_merged_selected.join(ch_discarded_reads)
- MINIMAP2(ch_contigs_and_discarded_reads, "03_filtering")
+ MINIMAP2(ch_contigs_and_discarded_reads, "03_filtering/03_2_reads_vs_filtered_assembly")
ch_bam_with_discarded_reads = MINIMAP2.out.bam
}
@@ -86,12 +86,12 @@ workflow STEP_03_ASSEMBLY_FILTER {
ch_final_bam = ch_bam_of_sample_without_discarded_contigs.mix(ch_merged_bam)
- GET_ALIGNMENT_METRICS(ch_final_bam, '03_filtering/')
+ GET_ALIGNMENT_METRICS(ch_final_bam, '03_filtering/03_2_reads_vs_filtered_assembly/')
ch_flagstat = GET_ALIGNMENT_METRICS.out.sam_flagstat
ch_coverage = GET_ALIGNMENT_METRICS.out.sam_coverage
- QUAST( ch_merged_selected, "03_filtering/quast_filtered" )
+ QUAST( ch_merged_selected, "03_filtering/03_1_filtered_assembly/assembly_metric" )
ch_quast_report = QUAST.out.report
emit:
diff --git a/subworkflows/05_alignment.nf b/subworkflows/05_protein_alignment.nf
similarity index 92%
rename from subworkflows/05_alignment.nf
rename to subworkflows/05_protein_alignment.nf
index 9017d47..56258c4 100644
--- a/subworkflows/05_alignment.nf
+++ b/subworkflows/05_protein_alignment.nf
@@ -1,6 +1,6 @@
include { DIAMOND } from '../modules/diamond'
-workflow STEP_05_ALIGNMENT {
+workflow STEP_05_PROTEIN_ALIGNMENT {
take:
prokka_faa
diamond
diff --git a/subworkflows/07_taxonomic_affi.nf b/subworkflows/07_taxonomic_affi.nf
index 8eb2640..99ead6c 100644
--- a/subworkflows/07_taxonomic_affi.nf
+++ b/subworkflows/07_taxonomic_affi.nf
@@ -25,9 +25,9 @@ workflow STEP_07_TAXO_AFFI {
PLOT_TAXONOMIC_AFFILIATIONS(ASSIGN_TAXONOMY.out.q_all.collect())
- KRONA_READS_COUNT(ASSIGN_TAXONOMY.out.krona_reads_count.collect(), "07_taxo_affi/plots", "krona_read_count_abundance.html")
+ KRONA_READS_COUNT(ASSIGN_TAXONOMY.out.krona_reads_count.collect(), "07_taxo_affi/07_3_plot", "krona_read_count_abundance.html")
- KRONA_DEPTH(ASSIGN_TAXONOMY.out.krona_depth.collect(), "07_taxo_affi/plots", "krona_mean_depth_abundance.html")
+ KRONA_DEPTH(ASSIGN_TAXONOMY.out.krona_depth.collect(), "07_taxo_affi/07_3_plot", "krona_mean_depth_abundance.html")
emit:
quantif_by_contig_lineage = QUANTIF_AND_TAXONOMIC_TABLE_CONTIGS.out.quantif_by_contig_lineage
--
GitLab
From 5b28724a97f1ffa0a486a02ffa27416608184858 Mon Sep 17 00:00:00 2001
From: DARBOT Vincent <vincent.darbot@inrae.fr>
Date: Fri, 25 Nov 2022 15:27:43 +0100
Subject: [PATCH 153/194] Update docs/output.md
---
docs/output.md | 17 ++++++++++++++++-
1 file changed, 16 insertions(+), 1 deletion(-)
diff --git a/docs/output.md b/docs/output.md
index 4ee8815..44af305 100644
--- a/docs/output.md
+++ b/docs/output.md
@@ -169,11 +169,26 @@ The `results/` directory contains a sub-directory for each step launched:
#### **08_binning/08_2_dereplicated_bins**
| File or directory/ | Description |
| ----------------------- | --------------------------------------- |
-| dereplicated_genomes/ | The dRep software allows to compare the bins of all samples in a pair-wise manner, and to clusturize the bins that share similar DNA content in terms of Average Nucleotide Identity (ANI). The threshold of 95% ANI is used to create a set of species-level representative genomes (SRGs). Within each cluster of bins, the best bin in terms of quality (completeness,contamination,N50,strain-heterogeneity) is selected as representative genome. The directory dereplicated_genomes contains all the representative bins that will be used in order to compare abundances of SRGs between the samples. |
+| dereplicated_genomes/ | The dRep software allows to compare the bins of all samples in a pair-wise manner, and to clusturize the bins that share similar DNA content in terms of Average Nucleotide Identity (ANI). The threshold of 95% ANI is used to create a set of species-level representative genomes (SRGs). Within each cluster of bins, the best bin in terms of quality (completeness,contamination,N50,strain-heterogeneity) is selected as representative genome. The directory dereplicated_genomes contains all the representative bins that will be used in order to compare abundances of SRGs between the samples. |
| data_tables/genomeInformation.csv | Calculated metrics of all the bins before de-replication, used to select the representative bin after de-replication. Completeness and contamination where calculated before with CheckM2, during bin_refinement step. dRep adds further metrics and use all four to choose the best bin. |
| data_tables/Bins_clusters_composition.tsv | Associates each representative bins cluster (1st column) to the bins list within the cluster (2nd column, bins seperates by commas). |
| figures/ | `Primary_clustering_dendrogram.pdf`: The primary clustering dendrogram summarizes the pair-wise Mash distance between all genomes in the genome list. The first clusturing used Mash, an incredibly fast but not robust algorithm, in order to accelerate the process during the second clusturing. **`Secondary_clustering_dendrograms.pdf`**: Each primary cluster with more than one member will have a page in the Secondary clustering dendrograms file. Bins with more than **95% of ANI distance similarity** (--drep_threshold default value paramater, that correspond to the threshold for separate species.) will be grouped together. **You can try different dRep thresholds to be more or less stringent, for example if the goal of dereplication is to generate a set of genomes that are distinct when mapping short reads, 98% ANI is an appropriate threshold.** You can also try higher threshold in order to separate species that belong to low divergent clades (96, or 97%). `Cluster_scoring.pdf`: Each secondary cluster will have its own page in the Cluster scoring figure. These figures show the score of each genome, as well as all metrics that can go into determin, and will always be the genome with the highest score.`Clustering scatterplots.pdf` provides some information about genome alignment statistics, and `Winning genomes.pdf` provides some information about only the “best†genomes of each replicate set, as well as a couple quick overall statistics.
+If you want to make further analysis about intra-population genetic diversity (microdiversity) on the genomes, you can use **inStrain** software specifically developed for this purpose. metagWGS outputs allows to easily run inStrain on this way:
+
+**1. Firstly, inStrain profile step calculates genome-level metrics for a genome of interest within each sample in which it is present:**
+
+`inStrain profile results/08_binning/08_4_mapping_on_final_bins/mapping/first_sample/first_sample.sort.bam results/08_binning/08_2_dereplicated_bins/dereplicated_genomes/first_bin.fa -o IS_output_first_sample`
+
+`inStrain profile results/08_binning/08_4_mapping_on_final_bins/mapping/first_sample/second_sample.sort.bam results/08_binning/08_2_dereplicated_bins/dereplicated_genomes/first_bin.fa -o IS_output_second_sample`
+
+`inStrain profile results/08_binning/08_4_mapping_on_final_bins/mapping/first_sample/third_sample.sort.bam results/08_binning/08_2_dereplicated_bins/dereplicated_genomes/first_bin.fa -o IS_output_third_sample`
+
+**2. Then, inStrain compare takes multiple inStrain profile objects (generated using the commands above) and performs strain-level comparisons :**
+
+`inStrain compare IS_output_first_sample IS_output_second_sample IS_output_third_sample`
+
+
#### **08_binning/08_3_gtdbtk/**
| File | Description |
| ----------------------- | --------------------------------------- |
--
GitLab
From 2948e1d3f433134850f9edf7e7646b5c75ba1619 Mon Sep 17 00:00:00 2001
From: Jean Mainguy <jean.mainguy@inra.fr>
Date: Fri, 25 Nov 2022 17:16:54 +0100
Subject: [PATCH 154/194] fix output organisation in metaflye process
---
modules/assembly.nf | 4 ++--
1 file changed, 2 insertions(+), 2 deletions(-)
diff --git a/modules/assembly.nf b/modules/assembly.nf
index 728e8b9..98cf3c6 100644
--- a/modules/assembly.nf
+++ b/modules/assembly.nf
@@ -98,9 +98,9 @@ process METAFLYE {
"""
mkdir metaflye_${meta.id}
- flye --pacbio-hifi $reads -o 'metaflye' --meta -t ${task.cpus} 2> metaflye_${meta.id}/${meta.id}.log
+ flye --pacbio-hifi $reads -o 'metaflye_${meta.id}' --meta -t ${task.cpus} 2> metaflye_${meta.id}/${meta.id}.log
- mv metaflye_${meta.id}/assembly.fasta metaflye_${meta.id}/${meta.id}.contigs.fa
+ mv metaflye_${meta.id}/assembly.fasta metaflye_${meta.id}/${meta.id}.contigs.fa
mv metaflye_${meta.id}/params.json metaflye_${meta.id}/${meta.id}.params.json
flye --version &> v_metaflye.txt
--
GitLab
From 1df91d299af41be92231cf4c31b6fb105c5bd949 Mon Sep 17 00:00:00 2001
From: Jean Mainguy <jean.mainguy@inra.fr>
Date: Fri, 25 Nov 2022 17:17:24 +0100
Subject: [PATCH 155/194] update output.md with new output organisation
---
docs/output.md | 149 +++++++++++++++++++++++++++++++------------------
1 file changed, 95 insertions(+), 54 deletions(-)
diff --git a/docs/output.md b/docs/output.md
index 44af305..080c01f 100644
--- a/docs/output.md
+++ b/docs/output.md
@@ -10,28 +10,30 @@ This document describes the output files produced by metagWGS.
| Directory/ | Description |
| ------------- | --------------------------------------- |
-| `work/` | Directory containing the Nextflow working files. Directory name can be changed if you use -w option of Nextflow. |
+| `work/` | Directory containing the Nextflow working files. Directory name can be changed if you use -w option of Nextflow. Once you are happy with the analysis and you do not intend to resume the pipeline execution, you can delete this directory. |
| `results/` | Directory containing metagWGS output files. Directory name can be changed if you use --outdir parameter of metagWGS. |
### Description of output files in `results/` directory:
The `results/` directory contains a sub-directory for each step launched:
-#### **01_clean_qc/01_1_cleaned_reads/**
+#### **01_clean_qc**
+
+##### **01_clean_qc/01_1_cleaned_reads/**
| File or directory/ | Description |
| ----------------------- | --------------------------------------- |
| `cleaned_SAMPLE_NAME_R{1,2}.fastq.gz` | There are one R1 and one R2 file for each sample. |
| `logs/` | Contains cutadapt (`SAMPLE_NAME_cutadapt.log`) and sickle (`SAMPLE_NAME_sickle.log`) log files for each sample. Only if you remove host reads, in `SAMPLE_NAME_cleaned_R{1,2}.nb_bases` you have the number of nucleotides into each cleaned R1 and R2 files of each sample. Only if you remove host reads, you also have a samtools flagstat file for each sample before removing host reads (`SAMPLE_NAME.no_filter.flagstat`) and into the directory `host_filter_flagstat/` there are the samtools flagstat files (`SAMPLE_NAME.host_filter.flagstat`) after removing host reads. |
-#### **01_clean_qc/01_2_qc/**
+##### **01_clean_qc/01_2_qc/**
| Directory/ | Description |
| ----------------------- | --------------------------------------- |
| `fastqc_raw/SAMPLE_NAME/` | Contains the results of fastQC on raw data: two files (`.html` and `.zip`) for R1 and two files for R2 (`.html` and `.zip`). |
| `fastqc_cleaned/cleaned_SAMPLE_NAME/` | Same as `01_2_qc/fastqc_raw/` but for cleaned data. |
-#### **01_clean_qc/01_3_taxonomic_affiliation_reads/**
+##### **01_clean_qc/01_3_taxonomic_affiliation_reads/**
| File | Description |
| ----------------------- | --------------------------------------- |
@@ -40,60 +42,86 @@ The `results/` directory contains a sub-directory for each step launched:
| `SAMPLE_NAME_MEM_verbose_only_classified.out.gz` | Compressed kaiju results. Each row corresponds to a classified reading. The first column 'C' indicates that the read is classified, the second column is the name of the read, the third is the NCBI taxon ID of the assigned taxon, the fourth is the length or score of the best match used for classification, the fifth is the taxon ID of all database sequences with the best match, the sixth is the accession number of all database sequences with the best match, and the last is the corresponding fragment sequence(s). |
| `match_length_kaiju_distribution.html` | Density plot of the size of the matches found by kaiju in reads of each sample. |
-#### **02_assembly/**
+#### **02_assembly**
-**NOTE:** in this directory you have either the results of assembly with `metaspades` or `megahit` but not both. You have chosen your assembly tool with `--assembly` parameter.
+#### **02_assembly/02_1_primary_assembly**
-| File or directory/ | Description |
-| ----------------------- | --------------------------------------- |
-| `metaspades/SAMPLE_NAME.log` | metaspades logs. Only if `--assembly "metaspades"` is used.|
-| `metaspades/SAMPLE_NAME.params.txt` | metaspades command line parameters used for the analysis. Only if `--assembly "metaspades"` is used.|
-| `metaspades/SAMPLE_NAME.contigs.fa` | metaspades assembly: nucleotide sequence of contigs. Only if `--assembly "metaspades"` is used.|
-| `megahit/SAMPLE_NAME.log` | megahit logs. Only if `--assembly "megahit"` is used.|
-| `megahit/SAMPLE_NAME.params.txt` | megahit options used for the analysis. Only if `--assembly "megahit"` is used.|
-| `megahit/SAMPLE_NAME.contigs.fa` | megahit assembly: nucleotide sequence of contigs. Only if `--assembly "megahit"` is used.|
-| `SAMPLE_NAME_all_contigs_QC/` | Contains metaQUAST quality control files of contigs. |
+**NOTE:** in this directory you have either the results of assembly with `metaspades` or `megahit` if you analyse short read data and `hifiasm-meta` or `metaflye` if you analyse HiFi data. You have chosen your assembly tool with `--assembly` parameter.
+
+| File or directory/ | Description |
+|--------------------------------|----------------------------------------------------------------------------------------------------------------------------------|
+| `SAMPLE_NAME.fna` | unfiltered assembly (primary assembly) with renamed contigs. Contig names follow this pattern: `<sample_name>_c<contig_number>`. |
+| `assembly_metric` | Contains metaQUAST quality control files of contigs. |
+| `<assembly_tool>_SAMPLE_NAME/` | output files of the assembly tool. It contains logs and original fasta files of the assembly before renaming. |
+
+
+
+##### **02_assembly/02_2_deduplicated_reads**
+
+**NOTE:** This directory contains deduplicated reads. It is created only for short read data.
+
+| File or directory/ | Description |
+|-------------------------------------|--------------------------------------------------------------|
| `SAMPLE_NAME_R{1,2}_dedup.fastq.gz` | Deduplicated reads (R1 and R2 files) for SAMPLE_NAME sample. |
-| `logs/` | Contains `SAMPLE_NAME.count_reads_on_contigs.flagstat`, `SAMPLE_NAME.count_reads_on_contigs.idxstats` and `SAMPLE_NAME_dedup_R{1,2}.nb_bases` files for each sample, generated after deduplication of reads. `SAMPLE_NAME.count_reads_on_contigs.flagstat` and `SAMPLE_NAME.count_reads_on_contigs.idxstats` are respectively the results of samtools flagstat (see informations [here](http://www.htslib.org/doc/samtools-flagstat.html)) and samtools idxstats (see description [here](http://www.htslib.org/doc/samtools-idxstats.html)), `SAMPLE_NAME_R{1,2}.nb_bases` corresponds to the number of nucleotides in the deduplicated R1 and R2 files. |
-#### **03_filtering/**
+##### **02_assembly/02_3_reads_vs_primary_assembly**
-| File or directory/ | Description |
-| ----------------------- | --------------------------------------- |
-| `SAMPLE_NAME_select_contigs_cpm[percent_identity].fasta` | Nucleotide sequence of contigs selected after filtering step with a percentage of identity of [percent_identity]. |
-| `SAMPLE_NAME_discard_contigs_cpm[percent_identity].fasta` | Nucleotide sequence of contigs discarded after filtering step with a percentage of identity of [percent_identity]. |
-| `SAMPLE_NAME_select_contigs_QC/` | Contains metaQUAST quality control files of filtered contigs. |
+**NOTE:** in this directory you have alignement metrics of reads aligned to the primary assembly (before the asssembly filering step if any). For short reads, reads have been deduplicated.
-#### **04_structural_annot/**
-| File | Description |
-| ----------------------- | --------------------------------------- |
-| `SAMPLE_NAME.annotated.faa` | Protein sequence of structural annotated genes. |
-| `SAMPLE_NAME.annotated.ffn` | Nucleotide sequence of structural annotated genes. |
-| `SAMPLE_NAME.annotated.fna` | Nucleotide sequence of contigs used by Prokka for the annotation of genes. In the used version of Prokka, it removes short contigs (<200bp). **WARNING:** these contigs are used in the following analysis. |
-| `SAMPLE_NAME.annotated.gff` | Coordinates of structural annotated genes into contigs. |
-| `SAMPLE_NAME_prot.len` | Length (in bp) of each gene annotated with Prokka |
+| File or directory/ | Description |
+|----------------------------------------|-------------------------------------------------------------------------------------------------|
+| `SAMPLE_NAME/SAMPLE_NAME.bam` | Samtools BAM file of sample reads align to the sample assembly. |
+| `SAMPLE_NAME/SAMPLE_NAME.coverage.tsv` | Samtools coverage file (see details [here](http://www.htslib.org/doc/samtools-coverage.html)) |
+| `SAMPLE_NAME/SAMPLE_NAME.flagstat` | Samtools flagstat file (see details [here](http://www.htslib.org/doc/samtools-flagstat.html) ) |
+| `SAMPLE_NAME/SAMPLE_NAME.idxstats` | Samtools idxstat file (see details [here](http://www.htslib.org/doc/samtools-flagstat.html) ) |
-**WARNING: starting from this step, the gene names follow this nomenclature: SAMPLE_NAME_CONTIG_ID.Prot_PROT_ID. Contig names follow the same nomenclature: SAMPLE_NAME_CONTIG_ID.**
-#### **05_alignment/05_1_reads_alignment_on_contigs/**
+#### **03_filtering**
-**WARNING:** contains a directory by sample. Each directory contains the following files:
+**NOTE:** This directory is created when the assembly filtering step is applied.
-| File | Description |
-| ----------------------- | --------------------------------------- |
-| `SAMPLE_NAME.sort.bam` | Alignment of reads on contigs (.bam file). |
-| `SAMPLE_NAME.sort.bam.bai` | Index of .bam file. |
-| `SAMPLE_NAME.sort.bam.idxstats` | Samtools idxstats file. See description [here](http://www.htslib.org/doc/samtools-idxstats.html). |
-| `SAMPLE_NAME_coverage.tsv` | Samtools coverage file. See description [here](http://www.htslib.org/doc/samtools-coverage.html). |
+##### **03_filtering/03_1_filtered_assembly/**
+
+| File or directory/ | Description |
+|------------------------------------------------------------------------|--------------------------------------------------------------------------------------------------------------|
+| `SAMPLE_NAME_select_contigs_cpm[cpm_threshold].fasta` | Selected contigs after filtering. This is the assembly fasta file used in the rest of the pipeline analysis. |
+| `discard_contigs/SAMPLE_NAME_discard_contigs_cpm[cpm_threshold].fasta` | Contigs that have been removed from the assembly. |
+| `assembly_metric/` | Contains metaQUAST quality control files of filtered assemblies.
+
+##### **03_filtering/03_2_reads_vs_filtered_assembly/**
+
+
+**NOTE:** This directory contains alignement metrics of reads aligned to the filtered assembly.
-#### **05_alignment/05_2_database_alignment/**
+
+| File or directory/ | Description |
+|----------------------------------------|-------------------------------------------------------------------------------------------------|
+| `SAMPLE_NAME/SAMPLE_NAME.bam` | Samtools BAM file of sample reads align to the sample assembly. |
+| `SAMPLE_NAME/SAMPLE_NAME.coverage.tsv` | Samtools coverage file (see details [here](http://www.htslib.org/doc/samtools-coverage.html)) |
+| `SAMPLE_NAME/SAMPLE_NAME.flagstat` | Samtools flagstat file (see details [here](http://www.htslib.org/doc/samtools-flagstat.html) ) |
+| `SAMPLE_NAME/SAMPLE_NAME.idxstats` | Samtools idxstat file (see details [here](http://www.htslib.org/doc/samtools-flagstat.html) ) |
+
+ |
+
+#### **04_structural_annot/**
+
+| File | Description |
+|-------------------------------|------------------------------------------------------------------|
+| `SAMPLE_NAME/SAMPLE_NAME.faa` | Fasta file of protein sequences of structural annotated genes. |
+| `SAMPLE_NAME/SAMPLE_NAME.ffn` | Fasta file of nucleotide sequence of structural annotated genes. |
+| `SAMPLE_NAME/SAMPLE_NAME.gff` | Coordinates of structural annotated genes into contigs. |
+| `SAMPLE_NAME/SAMPLE_NAME.txt` | Summary of the assembly structural annotation. |
+
+#### **05_protein_alignment/05_1_database_alignment/**
| File or directory/ | Description |
| ----------------------- | --------------------------------------- |
| `SAMPLE_NAME/SAMPLE_NAME_aln_diamond.m8` | Diamond results file. |
-#### **06_func_annot/06_1_clustering/**
+#### **06_func_annot**
+
+##### **06_func_annot/06_1_clustering/**
| File | Description |
| ----------------------- | --------------------------------------- |
@@ -104,7 +132,7 @@ The `results/` directory contains a sub-directory for each step launched:
| `All-cd-hit-est.[cd-hit percentage identity].fasta.clstr` | Text file of list of inter-sample clusters generated by cd-hit-est with [cd-hit percentage identity] percentage identity. |
| `table_clstr.txt` | Correspondance table of inter-sample clusters and intra-sample clusters. One line = one correspondance between an inter-sample cluster (first column) and an intra-sample cluster (second column). |
-#### **06_func_annot/06_2_quantification/**
+##### **06_func_annot/06_2_quantification/**
| File | Description |
| ----------------------- | --------------------------------------- |
@@ -114,7 +142,7 @@ The `results/` directory contains a sub-directory for each step launched:
| `Correspondence_global_clstr_contigs.txt` | Correspondance table of inter-sample clusters and initial genes. One line = one correspondance between an inter-sample cluster (first column) and an initial gene (second column). |
| `Clusters_Count_table_all_samples.txt` | Abundance table of reads. Each cell contains the sum of aligned reads on each initial gene of each inter-sample cluster for each sample (inter_sample clusters in rows and samples in colums).|
-#### **06_func_annot/06_3_functional_annotation/**
+##### **06_func_annot/06_3_functional_annotation/**
| File | Description |
| ----------------------- | --------------------------------------- |
@@ -130,6 +158,8 @@ The `results/` directory contains a sub-directory for each step launched:
#### **07_taxo_affi/**
+##### **07_taxo_affi/07_1_affiliation_per_sample**
+
| File | Description |
| ----------------------- | --------------------------------------- |
| `SAMPLE_NAME/SAMPLE_NAME.pergene.tsv` | Taxonomic affiliation of genes. One line corresponds to a gene (1st column), its corresponding taxon id (2nd column), its corresponding lineage (3rd column) and the tax ids of each level of this lineage (4th column). |
@@ -140,15 +170,23 @@ The `results/` directory contains a sub-directory for each step launched:
| `SAMPLE_NAME/SAMPLE_NAME_quantif_percontig_by_[taxonomic_level].tsv` | One file by taxonomic level (superkingdom, phylum, order, class, family, genus, species) for the sample `SAMPLE_NAME`. Quantification table of reads aligned on contigs affiliated to each lineage of the corresponding [taxonomic level]. One line = one taxonomic affiliation at this [taxonomic level] with is taxon id (1st column, `tax_id_by_level`), its lineage (2nd column, `lineage_by_level`), the name of contigs affiliated to this lineage (3rd column, `name_contigs`), the number of contigs affiliated to this lineage (4th column, `nb_contigs`), the sum of the number of reads aligned to these contigs (5th column, `nb_reads`) and the mean depth of these contigs (6th column, `depth`). |
| `SAMPLE_NAME/graphs/SAMPLE_NAME_aln_diamond.m8_contig_taxonomy_level.pdf` | Figure representing the number of contigs (y-axis) affiliated to each taxonomy levels (x-axis). |
| `SAMPLE_NAME/top_taxons_per_contig.tsv` | Details of top possible taxons per contig and for each taxonomic rank. For each rank, there is a list of the top possible taxons and their weigth in parentesis (only taxons with a weigth > 1 is written). For example in the genus column we can have `Escherichia (68.0);Shigella (20.0);Enterobacter (4.0);Salmonella (4.0);Felsduovirus (4.0)`. This file can be useful to understand an affiliation in detail. |
-| `SAMPLE_NAME/graphs/SAMPLE_NAME_aln_diamond.m8_contig_taxonomy_level.pdf` | Figure representing the number of contigs (y-axis) affiliated to each taxonomy levels (x-axis). |
-| `SAMPLE_NAME/graphs/SAMPLE_NAME_aln_diamond.m8_prot_taxonomy_level.pdf` | Figure representing the number of proteins (y-axis) affiliated to each taxonomy levels (x-axis). |
-| `SAMPLE_NAME/graphs/SAMPLE_NAME_aln_diamond.m8_nb_prot_annotated_and_assigned.pdf` | Figure representing the number of proteins (y-axis) in our contigs (`Total` bar), the number of proteins with a match into the database (`Annotated` bar) and the number of proteins with a match into the database which is found into the taxonomy (`Assigned` bar) (x-axis). |
+
+
+##### **07_taxo_affi/07_2_affiliation_merged**
+| File | Description |
+| ----------------------- | --------------------------------------- |
| `quantification_by_contig_lineage_all.tsv` | Quantification table of reads aligned on contigs affiliated to each lineage. One line = one taxonomic affiliation with its lineage (1st column, `lineage_by_level`), the taxon id at each level of this lineage (2nd column, `tax_id_by_level`), and then all next 3-columns blocks correspond to one sample. Each 3-column block corresponds to the name of contigs affiliated to this lineage (1st column, `name_contigs_SAMPLE_NAME_quantif_percontig`), the number of contigs affiliated to this lineage (2nd column, `nb_contigs_SAMPLE_NAME_quantif_percontig`), the sum of the number of reads aligned to these contigs (3rd column, `nb_reads_SAMPLE_NAME_quantif_percontig`) and the mean depth of these contigs (4th column, `depth_SAMPLE_NAME_quantif_percontig`). |
| `quantification_by_contig_lineage_[taxonomic_level].tsv` | One file by taxonomic level (superkingdom, phylum, order, class, family, genus, species). Quantification table of reads aligned on contigs affiliated to each lineage of the corresponding [taxonomic level]. One line = one taxonomic affiliation at this [taxonomic level] with its taxon id (1st column, `tax_id_by_level`), its lineage (2nd column, `lineage_by_level`), and then all next 3-columns blocks correspond to one sample. Each 3-column block corresponds to the name of contigs affiliated to this lineage (1st column, `name_contigs_SAMPLE_NAME_quantif_percontig_by_[taxonomic_level]`), the number of contigs affiliated to this lineage (2nd column, `nb_contigs_SAMPLE_NAME_quantif_percontig_by_[taxonomic_level]`) and the sum of the number of reads aligned to these contigs (3rd column, `nb_reads_SAMPLE_NAME_quantif_percontig_by_[taxonomic_level]`) and the mean depth of these contigs (4th column, `depth_SAMPLE_NAME_quantif_percontig_by_[taxonomic_level]`). |
-| `plots/krona_mean_depth_abundance.html` | Krona plot of the taxonomic affiliation of contigs. The abundance of a taxon is the percentage of the depth of the contigs affiliated to this taxon. |
-| `plots/krona_read_count_abundance.html` | Krona plot of the taxonomic affiliation of contigs. The abundance of a taxon is the percentage of reads mapping the contigs affiliated to this taxon. |
-| `plots/abundance_per_rank.html` | This plot represents the abundance of affiliation made for each taxonomic rank.|
-| `plots/most_abundant_taxa.html` | This plot represents the abundance of the 10 most abundant taxa at each taxonomic rank and for each sample.|
+
+
+##### **07_taxo_affi/07_3_plot/**
+
+| File | Description |
+| ----------------------- | --------------------------------------- |
+| `krona_mean_depth_abundance.html` | Krona plot of the taxonomic affiliation of contigs. The abundance of a taxon is the percentage of the depth of the contigs affiliated to this taxon. |
+| `krona_read_count_abundance.html` | Krona plot of the taxonomic affiliation of contigs. The abundance of a taxon is the percentage of reads mapping the contigs affiliated to this taxon. |
+| `abundance_per_rank.html` | This plot represents the abundance of affiliation made for each taxonomic rank.|
+| `most_abundant_taxa.html` | This plot represents the abundance of the 10 most abundant taxa at each taxonomic rank and for each sample.|
#### **08_binning/**
@@ -156,7 +194,10 @@ The `results/` directory contains a sub-directory for each step launched:
| ----------------------- | --------------------------------------- |
| `genomes_abundances.tsv` | Global informations about the final set of bins. One line = one bin id (1st column, `genome_id`), its bin name, retrieve from the lowest taxonomic rank affiliated by GTDB-Tk (2nd column, `genome_name`), the Domain, Phylum, Class, Order, Family, Genus, Species taxonomic affiliations made with GTDB-Tk (3rd, 4th, 5th, 6th, 7th, 8th and 9th columns), its quality metrics : `completeness` and `contamination`, retrieved with Checkm2 (10th and 11th columns), its size in bp (12th column, `genome_length`), its N50 metric (13th column, genome_N50), its number of contigs (14th column, `contig_count`). After that, there is 2 columns per sample : the number of reads and the mean depth associated to the bin within the specicic sample. Finally, the second to last column (`sum_numreads`) described the bin's total number of reads between all the samples, and the last (`mean_depth`) the average depth between all sample.|
-#### **08_binning/08_1_binning_per_sample**
+
+#### **08_binning**
+
+##### **08_binning/08_1_binning_per_sample**
| File or directory/ | Description |
| ----------------------- | --------------------------------------- |
@@ -166,7 +207,7 @@ The `results/` directory contains a sub-directory for each step launched:
| `SAMPLE_NAME/bin_refinement`| This directory contains bins fasta files from the aggregate binning module bin_refinement from metaWRAP tool, which takes into account the bins sets from the 3 individual binning tools to try to improve the quality of the bins (see informations [here](https://microbiomejournal.biomedcentral.com/articles/10.1186/s40168-018-0541-1)). |
| `SAMPLE_NAME/bin_refinement/unbinned_contigs.fasta`| Contigs from the sample assembly that are not retrieved in any bin. The proportion of non-binned contigs to the global size of the sample assembly can be seen in the MultiQC report, in the section Bins Size (bp) quality. |
-#### **08_binning/08_2_dereplicated_bins**
+##### **08_binning/08_2_dereplicated_bins**
| File or directory/ | Description |
| ----------------------- | --------------------------------------- |
| dereplicated_genomes/ | The dRep software allows to compare the bins of all samples in a pair-wise manner, and to clusturize the bins that share similar DNA content in terms of Average Nucleotide Identity (ANI). The threshold of 95% ANI is used to create a set of species-level representative genomes (SRGs). Within each cluster of bins, the best bin in terms of quality (completeness,contamination,N50,strain-heterogeneity) is selected as representative genome. The directory dereplicated_genomes contains all the representative bins that will be used in order to compare abundances of SRGs between the samples. |
@@ -189,12 +230,12 @@ If you want to make further analysis about intra-population genetic diversity (m
`inStrain compare IS_output_first_sample IS_output_second_sample IS_output_third_sample`
-#### **08_binning/08_3_gtdbtk/**
+##### **08_binning/08_3_gtdbtk/**
| File | Description |
| ----------------------- | --------------------------------------- |
| gtdbtk.bac120.summary.tsv | Taxonomic classifications provided by GTDB-Tk. One line = one bin id (1st column, `user_genome`), its taxonomical classification based on the closest reference genome from the GTDB-Tk database (2nd column, `classification`), the accession number of the closest reference genome (3rd column, `fastani_reference`). Please see GTDB-Tk documentation [here](https://ecogenomics.github.io/GTDBTk/files/summary.tsv.html) for information on additional columns.
-#### **08_4_mapping_on_final_bins**
+##### **08_4_mapping_on_final_bins**
| File | Description |
| ----------------------- | --------------------------------------- |
| mapping/SAMPLE_NAME/ | In order to compare genomes abundances between samples, mapping of metagenomics samples reads against the final set of de-replicated bins is performed.
--
GitLab
From 26700cb38773d9d730ae33c6fc26229c18a64dd7 Mon Sep 17 00:00:00 2001
From: Jean Mainguy <jean.mainguy@inra.fr>
Date: Fri, 25 Nov 2022 17:29:21 +0100
Subject: [PATCH 156/194] add table of content and update title size
---
docs/output.md | 104 ++++++++++++++++++++++++++++++++-----------------
1 file changed, 69 insertions(+), 35 deletions(-)
diff --git a/docs/output.md b/docs/output.md
index 080c01f..764a752 100644
--- a/docs/output.md
+++ b/docs/output.md
@@ -1,39 +1,74 @@
# metagWGS: Output
-## Introduction
-
This document describes the output files produced by metagWGS.
+- [metagWGS: Output](#metagwgs-output)
+ - [Description of output files and directories](#description-of-output-files-and-directories)
+ - [Description of output files in `results/` directory:](#description-of-output-files-in-results-directory)
+ - [**01\_clean\_qc**](#01_clean_qc)
+ - [**01\_clean\_qc/01\_1\_cleaned\_reads/**](#01_clean_qc01_1_cleaned_reads)
+ - [**01\_clean\_qc/01\_2\_qc/**](#01_clean_qc01_2_qc)
+ - [**01\_clean\_qc/01\_3\_taxonomic\_affiliation\_reads/**](#01_clean_qc01_3_taxonomic_affiliation_reads)
+ - [**02\_assembly**](#02_assembly)
+ - [**02\_assembly/02\_1\_primary\_assembly**](#02_assembly02_1_primary_assembly)
+ - [**02\_assembly/02\_2\_deduplicated\_reads**](#02_assembly02_2_deduplicated_reads)
+ - [**02\_assembly/02\_3\_reads\_vs\_primary\_assembly**](#02_assembly02_3_reads_vs_primary_assembly)
+ - [**03\_filtering**](#03_filtering)
+ - [**03\_filtering/03\_1\_filtered\_assembly/**](#03_filtering03_1_filtered_assembly)
+ - [**03\_filtering/03\_2\_reads\_vs\_filtered\_assembly/**](#03_filtering03_2_reads_vs_filtered_assembly)
+ - [**04\_structural\_annot/**](#04_structural_annot)
+ - [**05\_protein\_alignment/05\_1\_database\_alignment/**](#05_protein_alignment05_1_database_alignment)
+ - [**06\_func\_annot**](#06_func_annot)
+ - [**06\_func\_annot/06\_1\_clustering/**](#06_func_annot06_1_clustering)
+ - [**06\_func\_annot/06\_2\_quantification/**](#06_func_annot06_2_quantification)
+ - [**06\_func\_annot/06\_3\_functional\_annotation/**](#06_func_annot06_3_functional_annotation)
+ - [**07\_taxo\_affi/**](#07_taxo_affi)
+ - [**07\_taxo\_affi/07\_1\_affiliation\_per\_sample**](#07_taxo_affi07_1_affiliation_per_sample)
+ - [**07\_taxo\_affi/07\_2\_affiliation\_merged**](#07_taxo_affi07_2_affiliation_merged)
+ - [**07\_taxo\_affi/07\_3\_plot/**](#07_taxo_affi07_3_plot)
+ - [**08\_binning/**](#08_binning)
+ - [**08\_binning/08\_1\_binning\_per\_sample**](#08_binning08_1_binning_per_sample)
+ - [**08\_binning/08\_2\_dereplicated\_bins**](#08_binning08_2_dereplicated_bins)
+ - [**08\_binning/08\_3\_gtdbtk/**](#08_binning08_3_gtdbtk)
+ - [**08\_4\_mapping\_on\_final\_bins**](#08_4_mapping_on_final_bins)
+ - [**MultiQC/**](#multiqc)
+ - [**pipeline\_info/**](#pipeline_info)
+ - [Description of other files in your working directory (not in `results` directory):](#description-of-other-files-in-your-working-directory-not-in-results-directory)
+ - [Other files can be added to the working directory (not in `results` directory) if you use Nextflow specific options:](#other-files-can-be-added-to-the-working-directory-not-in-results-directory-if-you-use-nextflow-specific-options)
+
+
## Description of output files and directories
-### The pipeline will create the following folders in your working directory:
+The pipeline will create the following folders in your working directory:
| Directory/ | Description |
| ------------- | --------------------------------------- |
| `work/` | Directory containing the Nextflow working files. Directory name can be changed if you use -w option of Nextflow. Once you are happy with the analysis and you do not intend to resume the pipeline execution, you can delete this directory. |
| `results/` | Directory containing metagWGS output files. Directory name can be changed if you use --outdir parameter of metagWGS. |
-### Description of output files in `results/` directory:
+## Description of output files in `results/` directory:
The `results/` directory contains a sub-directory for each step launched:
-#### **01_clean_qc**
-##### **01_clean_qc/01_1_cleaned_reads/**
+
+### **01_clean_qc**
+
+#### **01_clean_qc/01_1_cleaned_reads/**
| File or directory/ | Description |
| ----------------------- | --------------------------------------- |
| `cleaned_SAMPLE_NAME_R{1,2}.fastq.gz` | There are one R1 and one R2 file for each sample. |
| `logs/` | Contains cutadapt (`SAMPLE_NAME_cutadapt.log`) and sickle (`SAMPLE_NAME_sickle.log`) log files for each sample. Only if you remove host reads, in `SAMPLE_NAME_cleaned_R{1,2}.nb_bases` you have the number of nucleotides into each cleaned R1 and R2 files of each sample. Only if you remove host reads, you also have a samtools flagstat file for each sample before removing host reads (`SAMPLE_NAME.no_filter.flagstat`) and into the directory `host_filter_flagstat/` there are the samtools flagstat files (`SAMPLE_NAME.host_filter.flagstat`) after removing host reads. |
-##### **01_clean_qc/01_2_qc/**
+#### **01_clean_qc/01_2_qc/**
| Directory/ | Description |
| ----------------------- | --------------------------------------- |
| `fastqc_raw/SAMPLE_NAME/` | Contains the results of fastQC on raw data: two files (`.html` and `.zip`) for R1 and two files for R2 (`.html` and `.zip`). |
| `fastqc_cleaned/cleaned_SAMPLE_NAME/` | Same as `01_2_qc/fastqc_raw/` but for cleaned data. |
-##### **01_clean_qc/01_3_taxonomic_affiliation_reads/**
+#### **01_clean_qc/01_3_taxonomic_affiliation_reads/**
| File | Description |
| ----------------------- | --------------------------------------- |
@@ -42,7 +77,7 @@ The `results/` directory contains a sub-directory for each step launched:
| `SAMPLE_NAME_MEM_verbose_only_classified.out.gz` | Compressed kaiju results. Each row corresponds to a classified reading. The first column 'C' indicates that the read is classified, the second column is the name of the read, the third is the NCBI taxon ID of the assigned taxon, the fourth is the length or score of the best match used for classification, the fifth is the taxon ID of all database sequences with the best match, the sixth is the accession number of all database sequences with the best match, and the last is the corresponding fragment sequence(s). |
| `match_length_kaiju_distribution.html` | Density plot of the size of the matches found by kaiju in reads of each sample. |
-#### **02_assembly**
+### **02_assembly**
#### **02_assembly/02_1_primary_assembly**
@@ -56,7 +91,7 @@ The `results/` directory contains a sub-directory for each step launched:
-##### **02_assembly/02_2_deduplicated_reads**
+#### **02_assembly/02_2_deduplicated_reads**
**NOTE:** This directory contains deduplicated reads. It is created only for short read data.
@@ -64,7 +99,7 @@ The `results/` directory contains a sub-directory for each step launched:
|-------------------------------------|--------------------------------------------------------------|
| `SAMPLE_NAME_R{1,2}_dedup.fastq.gz` | Deduplicated reads (R1 and R2 files) for SAMPLE_NAME sample. |
-##### **02_assembly/02_3_reads_vs_primary_assembly**
+#### **02_assembly/02_3_reads_vs_primary_assembly**
**NOTE:** in this directory you have alignement metrics of reads aligned to the primary assembly (before the asssembly filering step if any). For short reads, reads have been deduplicated.
@@ -77,11 +112,11 @@ The `results/` directory contains a sub-directory for each step launched:
| `SAMPLE_NAME/SAMPLE_NAME.idxstats` | Samtools idxstat file (see details [here](http://www.htslib.org/doc/samtools-flagstat.html) ) |
-#### **03_filtering**
+### **03_filtering**
**NOTE:** This directory is created when the assembly filtering step is applied.
-##### **03_filtering/03_1_filtered_assembly/**
+#### **03_filtering/03_1_filtered_assembly/**
| File or directory/ | Description |
|------------------------------------------------------------------------|--------------------------------------------------------------------------------------------------------------|
@@ -89,7 +124,7 @@ The `results/` directory contains a sub-directory for each step launched:
| `discard_contigs/SAMPLE_NAME_discard_contigs_cpm[cpm_threshold].fasta` | Contigs that have been removed from the assembly. |
| `assembly_metric/` | Contains metaQUAST quality control files of filtered assemblies.
-##### **03_filtering/03_2_reads_vs_filtered_assembly/**
+#### **03_filtering/03_2_reads_vs_filtered_assembly/**
**NOTE:** This directory contains alignement metrics of reads aligned to the filtered assembly.
@@ -104,7 +139,7 @@ The `results/` directory contains a sub-directory for each step launched:
|
-#### **04_structural_annot/**
+### **04_structural_annot/**
| File | Description |
|-------------------------------|------------------------------------------------------------------|
@@ -113,15 +148,15 @@ The `results/` directory contains a sub-directory for each step launched:
| `SAMPLE_NAME/SAMPLE_NAME.gff` | Coordinates of structural annotated genes into contigs. |
| `SAMPLE_NAME/SAMPLE_NAME.txt` | Summary of the assembly structural annotation. |
-#### **05_protein_alignment/05_1_database_alignment/**
+### **05_protein_alignment/05_1_database_alignment/**
| File or directory/ | Description |
| ----------------------- | --------------------------------------- |
| `SAMPLE_NAME/SAMPLE_NAME_aln_diamond.m8` | Diamond results file. |
-#### **06_func_annot**
+### **06_func_annot**
-##### **06_func_annot/06_1_clustering/**
+#### **06_func_annot/06_1_clustering/**
| File | Description |
| ----------------------- | --------------------------------------- |
@@ -132,7 +167,7 @@ The `results/` directory contains a sub-directory for each step launched:
| `All-cd-hit-est.[cd-hit percentage identity].fasta.clstr` | Text file of list of inter-sample clusters generated by cd-hit-est with [cd-hit percentage identity] percentage identity. |
| `table_clstr.txt` | Correspondance table of inter-sample clusters and intra-sample clusters. One line = one correspondance between an inter-sample cluster (first column) and an intra-sample cluster (second column). |
-##### **06_func_annot/06_2_quantification/**
+#### **06_func_annot/06_2_quantification/**
| File | Description |
| ----------------------- | --------------------------------------- |
@@ -142,7 +177,7 @@ The `results/` directory contains a sub-directory for each step launched:
| `Correspondence_global_clstr_contigs.txt` | Correspondance table of inter-sample clusters and initial genes. One line = one correspondance between an inter-sample cluster (first column) and an initial gene (second column). |
| `Clusters_Count_table_all_samples.txt` | Abundance table of reads. Each cell contains the sum of aligned reads on each initial gene of each inter-sample cluster for each sample (inter_sample clusters in rows and samples in colums).|
-##### **06_func_annot/06_3_functional_annotation/**
+#### **06_func_annot/06_3_functional_annotation/**
| File | Description |
| ----------------------- | --------------------------------------- |
@@ -156,9 +191,9 @@ The `results/` directory contains a sub-directory for each step launched:
| `KEGG_Module_abundance.tsv` | Quantification table storing for each KEGG_Module (rows) the sum of aligned reads into all genes having this functional annotation for each sample (columns). |
| `PFAM_abundance.tsv` | Quantification table storing for each PFAM (rows) the sum of aligned reads into all genes having this functional annotation for each sample (columns). |
-#### **07_taxo_affi/**
+### **07_taxo_affi/**
-##### **07_taxo_affi/07_1_affiliation_per_sample**
+#### **07_taxo_affi/07_1_affiliation_per_sample**
| File | Description |
| ----------------------- | --------------------------------------- |
@@ -172,14 +207,15 @@ The `results/` directory contains a sub-directory for each step launched:
| `SAMPLE_NAME/top_taxons_per_contig.tsv` | Details of top possible taxons per contig and for each taxonomic rank. For each rank, there is a list of the top possible taxons and their weigth in parentesis (only taxons with a weigth > 1 is written). For example in the genus column we can have `Escherichia (68.0);Shigella (20.0);Enterobacter (4.0);Salmonella (4.0);Felsduovirus (4.0)`. This file can be useful to understand an affiliation in detail. |
-##### **07_taxo_affi/07_2_affiliation_merged**
+#### **07_taxo_affi/07_2_affiliation_merged**
+
| File | Description |
| ----------------------- | --------------------------------------- |
| `quantification_by_contig_lineage_all.tsv` | Quantification table of reads aligned on contigs affiliated to each lineage. One line = one taxonomic affiliation with its lineage (1st column, `lineage_by_level`), the taxon id at each level of this lineage (2nd column, `tax_id_by_level`), and then all next 3-columns blocks correspond to one sample. Each 3-column block corresponds to the name of contigs affiliated to this lineage (1st column, `name_contigs_SAMPLE_NAME_quantif_percontig`), the number of contigs affiliated to this lineage (2nd column, `nb_contigs_SAMPLE_NAME_quantif_percontig`), the sum of the number of reads aligned to these contigs (3rd column, `nb_reads_SAMPLE_NAME_quantif_percontig`) and the mean depth of these contigs (4th column, `depth_SAMPLE_NAME_quantif_percontig`). |
| `quantification_by_contig_lineage_[taxonomic_level].tsv` | One file by taxonomic level (superkingdom, phylum, order, class, family, genus, species). Quantification table of reads aligned on contigs affiliated to each lineage of the corresponding [taxonomic level]. One line = one taxonomic affiliation at this [taxonomic level] with its taxon id (1st column, `tax_id_by_level`), its lineage (2nd column, `lineage_by_level`), and then all next 3-columns blocks correspond to one sample. Each 3-column block corresponds to the name of contigs affiliated to this lineage (1st column, `name_contigs_SAMPLE_NAME_quantif_percontig_by_[taxonomic_level]`), the number of contigs affiliated to this lineage (2nd column, `nb_contigs_SAMPLE_NAME_quantif_percontig_by_[taxonomic_level]`) and the sum of the number of reads aligned to these contigs (3rd column, `nb_reads_SAMPLE_NAME_quantif_percontig_by_[taxonomic_level]`) and the mean depth of these contigs (4th column, `depth_SAMPLE_NAME_quantif_percontig_by_[taxonomic_level]`). |
-##### **07_taxo_affi/07_3_plot/**
+#### **07_taxo_affi/07_3_plot/**
| File | Description |
| ----------------------- | --------------------------------------- |
@@ -188,16 +224,14 @@ The `results/` directory contains a sub-directory for each step launched:
| `abundance_per_rank.html` | This plot represents the abundance of affiliation made for each taxonomic rank.|
| `most_abundant_taxa.html` | This plot represents the abundance of the 10 most abundant taxa at each taxonomic rank and for each sample.|
-#### **08_binning/**
+### **08_binning/**
| File | Description |
| ----------------------- | --------------------------------------- |
| `genomes_abundances.tsv` | Global informations about the final set of bins. One line = one bin id (1st column, `genome_id`), its bin name, retrieve from the lowest taxonomic rank affiliated by GTDB-Tk (2nd column, `genome_name`), the Domain, Phylum, Class, Order, Family, Genus, Species taxonomic affiliations made with GTDB-Tk (3rd, 4th, 5th, 6th, 7th, 8th and 9th columns), its quality metrics : `completeness` and `contamination`, retrieved with Checkm2 (10th and 11th columns), its size in bp (12th column, `genome_length`), its N50 metric (13th column, genome_N50), its number of contigs (14th column, `contig_count`). After that, there is 2 columns per sample : the number of reads and the mean depth associated to the bin within the specicic sample. Finally, the second to last column (`sum_numreads`) described the bin's total number of reads between all the samples, and the last (`mean_depth`) the average depth between all sample.|
-#### **08_binning**
-
-##### **08_binning/08_1_binning_per_sample**
+#### **08_binning/08_1_binning_per_sample**
| File or directory/ | Description |
| ----------------------- | --------------------------------------- |
@@ -207,7 +241,7 @@ The `results/` directory contains a sub-directory for each step launched:
| `SAMPLE_NAME/bin_refinement`| This directory contains bins fasta files from the aggregate binning module bin_refinement from metaWRAP tool, which takes into account the bins sets from the 3 individual binning tools to try to improve the quality of the bins (see informations [here](https://microbiomejournal.biomedcentral.com/articles/10.1186/s40168-018-0541-1)). |
| `SAMPLE_NAME/bin_refinement/unbinned_contigs.fasta`| Contigs from the sample assembly that are not retrieved in any bin. The proportion of non-binned contigs to the global size of the sample assembly can be seen in the MultiQC report, in the section Bins Size (bp) quality. |
-##### **08_binning/08_2_dereplicated_bins**
+#### **08_binning/08_2_dereplicated_bins**
| File or directory/ | Description |
| ----------------------- | --------------------------------------- |
| dereplicated_genomes/ | The dRep software allows to compare the bins of all samples in a pair-wise manner, and to clusturize the bins that share similar DNA content in terms of Average Nucleotide Identity (ANI). The threshold of 95% ANI is used to create a set of species-level representative genomes (SRGs). Within each cluster of bins, the best bin in terms of quality (completeness,contamination,N50,strain-heterogeneity) is selected as representative genome. The directory dereplicated_genomes contains all the representative bins that will be used in order to compare abundances of SRGs between the samples. |
@@ -230,38 +264,38 @@ If you want to make further analysis about intra-population genetic diversity (m
`inStrain compare IS_output_first_sample IS_output_second_sample IS_output_third_sample`
-##### **08_binning/08_3_gtdbtk/**
+#### **08_binning/08_3_gtdbtk/**
| File | Description |
| ----------------------- | --------------------------------------- |
| gtdbtk.bac120.summary.tsv | Taxonomic classifications provided by GTDB-Tk. One line = one bin id (1st column, `user_genome`), its taxonomical classification based on the closest reference genome from the GTDB-Tk database (2nd column, `classification`), the accession number of the closest reference genome (3rd column, `fastani_reference`). Please see GTDB-Tk documentation [here](https://ecogenomics.github.io/GTDBTk/files/summary.tsv.html) for information on additional columns.
-##### **08_4_mapping_on_final_bins**
+#### **08_4_mapping_on_final_bins**
| File | Description |
| ----------------------- | --------------------------------------- |
| mapping/SAMPLE_NAME/ | In order to compare genomes abundances between samples, mapping of metagenomics samples reads against the final set of de-replicated bins is performed.
| mapping/SAMPLE_NAME/`SAMPLE_NAME.sort.bam` | Alignment of reads on contigs (.bam file). |
| mapping/SAMPLE_NAME/`SAMPLE_NAME.sort.bam.bai` | Index of .bam file. |
-#### **MultiQC/**
+### **MultiQC/**
| File | Description |
| ----------------------- | --------------------------------------- |
| `multiqc_report.html` | MultiQC report file containing graphs and a summary of analysis done by metagWGS. |
-#### **pipeline_info/**
+### **pipeline_info/**
| File | Description |
| ----------------------- | --------------------------------------- |
| `software_versions.csv` | Indicates the versions of the tools used in the pipeline. |
| `db_versions.tsv` | Indicates the size, date of last modification and path of the file or folder for each databank used in the pipeline. For the host genome, the number of sequence is in parenthesis with the size of the file. |
-### Description of other files in your working directory (not in `results` directory):
+## Description of other files in your working directory (not in `results` directory):
| File | Description |
| ------------- | ---------------------- |
| `.nextflow_log` | Log file from Nextflow. |
-### Other files can be added to the working directory (not in `results` directory) if you use Nextflow specific options:
+## Other files can be added to the working directory (not in `results` directory) if you use Nextflow specific options:
| Option | File | Description |
| ------------- | --------------- | ---------------------- |
--
GitLab
From a6752ea07b0c3c2f8eb9d36b8979a9df7d538a97 Mon Sep 17 00:00:00 2001
From: Jean Mainguy <jean.mainguy@inra.fr>
Date: Wed, 30 Nov 2022 10:48:25 +0100
Subject: [PATCH 157/194] remove typo making script fail
---
bin/format_bins_stat_to_multiqc.py | 2 +-
1 file changed, 1 insertion(+), 1 deletion(-)
diff --git a/bin/format_bins_stat_to_multiqc.py b/bin/format_bins_stat_to_multiqc.py
index 05fdeb6..cefd801 100755
--- a/bin/format_bins_stat_to_multiqc.py
+++ b/bin/format_bins_stat_to_multiqc.py
@@ -22,7 +22,7 @@ import pandas as pd
def parse_arguments():
- """Parse script arguments."""bins_per_sample_summarize.py
+ """Parse script arguments."""
parser = ArgumentParser(description="...",
formatter_class=ArgumentDefaultsHelpFormatter)
--
GitLab
From 28c2d0046916edcc59cbfd84999e61ca6915ccf7 Mon Sep 17 00:00:00 2001
From: DARBOT Vincent <vincent.darbot@inrae.fr>
Date: Fri, 2 Dec 2022 14:06:57 +0100
Subject: [PATCH 158/194] Update docs/output.md
---
docs/output.md | 14 +++++---------
1 file changed, 5 insertions(+), 9 deletions(-)
diff --git a/docs/output.md b/docs/output.md
index 764a752..f2bd2e4 100644
--- a/docs/output.md
+++ b/docs/output.md
@@ -249,19 +249,15 @@ The `results/` directory contains a sub-directory for each step launched:
| data_tables/Bins_clusters_composition.tsv | Associates each representative bins cluster (1st column) to the bins list within the cluster (2nd column, bins seperates by commas). |
| figures/ | `Primary_clustering_dendrogram.pdf`: The primary clustering dendrogram summarizes the pair-wise Mash distance between all genomes in the genome list. The first clusturing used Mash, an incredibly fast but not robust algorithm, in order to accelerate the process during the second clusturing. **`Secondary_clustering_dendrograms.pdf`**: Each primary cluster with more than one member will have a page in the Secondary clustering dendrograms file. Bins with more than **95% of ANI distance similarity** (--drep_threshold default value paramater, that correspond to the threshold for separate species.) will be grouped together. **You can try different dRep thresholds to be more or less stringent, for example if the goal of dereplication is to generate a set of genomes that are distinct when mapping short reads, 98% ANI is an appropriate threshold.** You can also try higher threshold in order to separate species that belong to low divergent clades (96, or 97%). `Cluster_scoring.pdf`: Each secondary cluster will have its own page in the Cluster scoring figure. These figures show the score of each genome, as well as all metrics that can go into determin, and will always be the genome with the highest score.`Clustering scatterplots.pdf` provides some information about genome alignment statistics, and `Winning genomes.pdf` provides some information about only the “best†genomes of each replicate set, as well as a couple quick overall statistics.
-If you want to make further analysis about intra-population genetic diversity (microdiversity) on the genomes, you can use **inStrain** software specifically developed for this purpose. metagWGS outputs allows to easily run inStrain on this way:
+If you want to make further analysis about intra-population genetic diversity (microdiversity) on the genomes, you can use **inStrain** software specifically developed for this purpose. The documentation is here:
-**1. Firstly, inStrain profile step calculates genome-level metrics for a genome of interest within each sample in which it is present:**
+https://instrain.readthedocs.io/en/latest/tutorial.html#quick-start
-`inStrain profile results/08_binning/08_4_mapping_on_final_bins/mapping/first_sample/first_sample.sort.bam results/08_binning/08_2_dereplicated_bins/dereplicated_genomes/first_bin.fa -o IS_output_first_sample`
+ metagWGS outputs allows to easily run inStrain and are available here :
-`inStrain profile results/08_binning/08_4_mapping_on_final_bins/mapping/first_sample/second_sample.sort.bam results/08_binning/08_2_dereplicated_bins/dereplicated_genomes/first_bin.fa -o IS_output_second_sample`
+ - bam files : results/08_binning/08_4_mapping_on_final_bins/mapping/first_sample/first_sample.sort.bam
-`inStrain profile results/08_binning/08_4_mapping_on_final_bins/mapping/first_sample/third_sample.sort.bam results/08_binning/08_2_dereplicated_bins/dereplicated_genomes/first_bin.fa -o IS_output_third_sample`
-
-**2. Then, inStrain compare takes multiple inStrain profile objects (generated using the commands above) and performs strain-level comparisons :**
-
-`inStrain compare IS_output_first_sample IS_output_second_sample IS_output_third_sample`
+ - bin fasta files : results/08_binning/08_2_dereplicated_bins/dereplicated_genomes/first_bin.fa
#### **08_binning/08_3_gtdbtk/**
--
GitLab
From be39d42075700b54fed771ba42bc03a3a72f7c84 Mon Sep 17 00:00:00 2001
From: DARBOT Vincent <vincent.darbot@inrae.fr>
Date: Fri, 2 Dec 2022 14:49:29 +0100
Subject: [PATCH 159/194] Update assets/multiqc/barplot_bin_size_header.txt
---
assets/multiqc/barplot_bin_size_header.txt | 2 +-
1 file changed, 1 insertion(+), 1 deletion(-)
diff --git a/assets/multiqc/barplot_bin_size_header.txt b/assets/multiqc/barplot_bin_size_header.txt
index d8f5b90..2e9ece6 100644
--- a/assets/multiqc/barplot_bin_size_header.txt
+++ b/assets/multiqc/barplot_bin_size_header.txt
@@ -1,6 +1,6 @@
# id: 'bins_quality_size'
# section_name: 'Bins Size (bp) quality'
-# description: 'Number of sequences by quality category (according to the bins quality category in the figure above), according to MIMAG (Minimum information about a metagenome-assembled genome) standards.'
+# description: 'Number of sequences by quality category (according to the bins quality category in the figure above), according to MIMAG (Minimum information about a metagenome-assembled genome) standards. The "not-binned" part refers to the number of reads that are not aligned on any bin. The x-axis corresponds to the number of sequences (or proportion), the y-axis indicates the samples.'
# format: 'tsv'
# plot_type: 'bargraph'
# categories:
--
GitLab
From ba6ad5b209344791d67e116227ef70eb8f6984b1 Mon Sep 17 00:00:00 2001
From: Jean Mainguy <jean.mainguy@inra.fr>
Date: Fri, 2 Dec 2022 15:13:56 +0100
Subject: [PATCH 160/194] fix bam header to have a coherent header with the
final assembly
---
modules/read_alignment_manipulation.nf | 17 ++++++++++++-----
1 file changed, 12 insertions(+), 5 deletions(-)
diff --git a/modules/read_alignment_manipulation.nf b/modules/read_alignment_manipulation.nf
index ca0fe5d..c5a67e4 100644
--- a/modules/read_alignment_manipulation.nf
+++ b/modules/read_alignment_manipulation.nf
@@ -89,16 +89,23 @@ process MERGE_BAM_FILES {
tag "${meta.id}"
input:
- tuple val(meta), path(bam1), path(bam2)
+ tuple val(meta), path(bam_discarded_reads), path(bam_selected_contigs)
output:
- tuple val(meta), path("${meta.id}.filtered.bam"), path("${meta.id}.filtered.bam.bai") , emit: bam
+ tuple val(meta), path("${meta.id}.filtered.sort.bam"), path("${meta.id}.filtered.sort.bam.bai") , emit: bam
script:
- """
- samtools merge -@ ${task.cpus} -o ${meta.id}.filtered.bam $bam1 $bam2
- samtools index -@ ${task.cpus} ${meta.id}.filtered.bam
+ """
+ samtools merge -@ ${task.cpus} $bam_discarded_reads $bam_selected_contigs -o merged.bam
+
+ # reheader the bam to get only header corresponding to the final assembly only
+ samtools view -H $bam_discarded_reads > filtered_assembly.header
+ samtools reheader filtered_assembly.header merged.bam | samtools sort -@ ${task.cpus} -o ${meta.id}.filtered.sort.bam
+
+ samtools index -@ ${task.cpus} ${meta.id}.filtered.sort.bam
+
+ rm merged.bam
"""
}
--
GitLab
From 807f1ebbd77c749946b1f1da4a430e4e58ecb33e Mon Sep 17 00:00:00 2001
From: DARBOT Vincent <vincent.darbot@inrae.fr>
Date: Fri, 2 Dec 2022 15:22:22 +0100
Subject: [PATCH 161/194] Update README.md
---
README.md | 2 +-
1 file changed, 1 insertion(+), 1 deletion(-)
diff --git a/README.md b/README.md
index cf0d150..595160f 100644
--- a/README.md
+++ b/README.md
@@ -21,7 +21,7 @@ Many of these steps are optional and their necessity depends on the desired anal
* `S02_ASSEMBLY`
* assembles reads ([metaSPAdes](https://github.com/ablab/spades) or [Megahit](https://github.com/voutcn/megahit) or [Hifiasm_meta](https://github.com/lh3/hifiasm-meta) or [metaFlye](https://github.com/fenderglass/Flye))
* assesses the quality of assembly ([metaQUAST](http://quast.sourceforge.net/metaquast))
- * deduplicates reads ([BWA-MEM2](https://github.com/bwa-mem2/bwa-mem2) or [Minimap2](https://github.com/lh3/minimap2) + [Samtools](http://www.htslib.org/))
+ * reads deduplication ([BWA-MEM2](https://github.com/bwa-mem2/bwa-mem2) or [Minimap2](https://github.com/lh3/minimap2) + [Samtools](http://www.htslib.org/))
* `S03_FILTERING`
* filters contigs with low CPM value ([Filter_contig_per_cpm.py](bin/Filter_contig_per_cpm.py) + [metaQUAST](http://quast.sourceforge.net/metaquast))
* `S04_STRUCTURAL_ANNOT`
--
GitLab
From 3a41ab60427c92888d13e40d3813d07ff40e0702 Mon Sep 17 00:00:00 2001
From: Claire Hoede <claire.hoede@inra.fr>
Date: Wed, 14 Dec 2022 07:39:53 +0100
Subject: [PATCH 162/194] accent warning in path or file name
---
docs/usage.md | 2 ++
1 file changed, 2 insertions(+)
diff --git a/docs/usage.md b/docs/usage.md
index d0b1a4c..ed48bf6 100644
--- a/docs/usage.md
+++ b/docs/usage.md
@@ -46,6 +46,8 @@ See [dedicated section](usage.md#4-samplesheet) for more information.
## II. Input files
+**WARNING:** Do not use any accent in any path or file name.
+
### 1. General mandatory files
Launching metagWGS involves the use of mandatory files:
--
GitLab
From a96618ead70a5911426066ef841ab196d384fce3 Mon Sep 17 00:00:00 2001
From: Vincent Darbot <vincent.darbot@inrae.fr>
Date: Thu, 15 Dec 2022 11:51:46 +0100
Subject: [PATCH 163/194] modify legend for barplot_bin_size_header.txt
---
assets/multiqc/barplot_bin_size_header.txt | 2 +-
1 file changed, 1 insertion(+), 1 deletion(-)
diff --git a/assets/multiqc/barplot_bin_size_header.txt b/assets/multiqc/barplot_bin_size_header.txt
index 2e9ece6..90ffe86 100644
--- a/assets/multiqc/barplot_bin_size_header.txt
+++ b/assets/multiqc/barplot_bin_size_header.txt
@@ -1,6 +1,6 @@
# id: 'bins_quality_size'
# section_name: 'Bins Size (bp) quality'
-# description: 'Number of sequences by quality category (according to the bins quality category in the figure above), according to MIMAG (Minimum information about a metagenome-assembled genome) standards. The "not-binned" part refers to the number of reads that are not aligned on any bin. The x-axis corresponds to the number of sequences (or proportion), the y-axis indicates the samples.'
+# description: 'Cumulative length of sequences by quality category (according to the bins quality category in the figure above), according to MIMAG (Minimum information about a metagenome-assembled genome) standards. The "not-binned" part refers to the number of reads that are not aligned on any bin. The x-axis corresponds to the number of sequences (or proportion), the y-axis indicates the samples.'
# format: 'tsv'
# plot_type: 'bargraph'
# categories:
--
GitLab
From 4e3a80d838a8fade0b1083754183e5db109d967b Mon Sep 17 00:00:00 2001
From: Vincent Darbot <vincent.darbot@inrae.fr>
Date: Fri, 16 Dec 2022 12:03:05 +0100
Subject: [PATCH 164/194] rights access to merge files
---
bin/merge_abundance_and_functional_annotations.py | 0
bin/merge_annotations.py | 0
2 files changed, 0 insertions(+), 0 deletions(-)
mode change 100644 => 100755 bin/merge_abundance_and_functional_annotations.py
mode change 100644 => 100755 bin/merge_annotations.py
diff --git a/bin/merge_abundance_and_functional_annotations.py b/bin/merge_abundance_and_functional_annotations.py
old mode 100644
new mode 100755
diff --git a/bin/merge_annotations.py b/bin/merge_annotations.py
old mode 100644
new mode 100755
--
GitLab
From 56d46390913b71f1f8b0e4568cc4dc99487983c5 Mon Sep 17 00:00:00 2001
From: Jean Mainguy <jean.mainguy@inra.fr>
Date: Fri, 16 Dec 2022 17:15:17 +0100
Subject: [PATCH 165/194] realign after filtering except when filtering has no
effect
---
docs/output.md | 8 ++-
main.nf | 8 ++-
modules/filtering_cpm.nf | 5 +-
modules/read_alignment_manipulation.nf | 86 --------------------------
subworkflows/03_filtering.nf | 71 ++++++++++++---------
5 files changed, 56 insertions(+), 122 deletions(-)
diff --git a/docs/output.md b/docs/output.md
index f2bd2e4..f10e72a 100644
--- a/docs/output.md
+++ b/docs/output.md
@@ -116,7 +116,7 @@ The `results/` directory contains a sub-directory for each step launched:
**NOTE:** This directory is created when the assembly filtering step is applied.
-#### **03_filtering/03_1_filtered_assembly/**
+#### **03_filtering/filtering_at_<cpm_threshold>cpm/03_1_filtered_assembly/**
| File or directory/ | Description |
|------------------------------------------------------------------------|--------------------------------------------------------------------------------------------------------------|
@@ -124,10 +124,12 @@ The `results/` directory contains a sub-directory for each step launched:
| `discard_contigs/SAMPLE_NAME_discard_contigs_cpm[cpm_threshold].fasta` | Contigs that have been removed from the assembly. |
| `assembly_metric/` | Contains metaQUAST quality control files of filtered assemblies.
-#### **03_filtering/03_2_reads_vs_filtered_assembly/**
+#### **03_filtering/filtering_at_<cpm_threshold>cpm/03_2_reads_vs_filtered_assembly/**
-**NOTE:** This directory contains alignement metrics of reads aligned to the filtered assembly.
+**NOTE:**
+- This directory contains alignement metrics of reads aligned to the filtered assembly.
+- When the filtering step has no effect on the assembly (no contig are filtered out), the bam of the reads over the assembly does not change with the one from of this assembly tool `02_assembly/02_3_reads_vs_primary_assembly` adn consequently the bam found here will be a symbolic link with the bam from the assembly file.
| File or directory/ | Description |
diff --git a/main.nf b/main.nf
index d9bc02b..c46f90b 100644
--- a/main.nf
+++ b/main.nf
@@ -427,19 +427,23 @@ workflow {
S03_FILTERING (
ch_assembly,
+ ch_preprocessed_reads,
ch_idxstats,
ch_bam,
- ch_min_contigs_cpm
+ ch_min_contigs_cpm,
+
)
ch_assembly = S03_FILTERING.out.selected_contigs
ch_bam = S03_FILTERING.out.bam
- ch_sam_coverage = S03_FILTERING.out.sam_coverage
+
ch_quast = S03_FILTERING.out.quast_report
ch_quast_after_filter_report = ch_quast.map{ meta, quast_report -> quast_report }
+
+ ch_sam_coverage = S03_FILTERING.out.sam_coverage
ch_final_assembly_flagstat_report = S03_FILTERING.out.sam_flagstat
}
diff --git a/modules/filtering_cpm.nf b/modules/filtering_cpm.nf
index 5dd2c90..5c9060d 100644
--- a/modules/filtering_cpm.nf
+++ b/modules/filtering_cpm.nf
@@ -20,13 +20,14 @@ process MERGE_ASSEMBLY_FILTER {
label 'ASSEMBLY_FILTER'
tag "${meta.id}"
- publishDir "${params.outdir}/03_filtering/03_1_filtered_assembly/", mode: 'copy', pattern: "*_select_contigs*"
- publishDir "${params.outdir}/03_filtering/03_1_filtered_assembly/discard_contigs", mode: 'copy', pattern: "*_discard_contigs*"
+ publishDir "${params.outdir}/${publishDir_path}/", mode: 'copy', pattern: "*_select_contigs*"
+ publishDir "${params.outdir}/${publishDir_path}/discard_contigs", mode: 'copy', pattern: "*_discard_contigs*"
input:
tuple val(meta), path(select_fasta)
tuple val(meta), path(discard_fasta)
val min_cpm
+ val(publishDir_path)
output:
tuple val(meta), path("${meta.id}_select_contigs_cpm${min_cpm}.fasta"), emit: merged_selected
diff --git a/modules/read_alignment_manipulation.nf b/modules/read_alignment_manipulation.nf
index c5a67e4..858bf81 100644
--- a/modules/read_alignment_manipulation.nf
+++ b/modules/read_alignment_manipulation.nf
@@ -23,89 +23,3 @@ process GET_ALIGNMENT_METRICS {
"""
}
-
-process FILTER_BAM {
- tag "${meta.id}"
-
- input:
- tuple val(meta), path(discarded_contigs), path(bam), path(bai)
-
- output:
- tuple val(meta), path("selected_contigs.bam"), emit: selected_contigs_bam
- tuple val(meta), path("discarded_contigs.bam"), emit: discarded_contigs_bam
-
-
- script:
- """
-
- samtools faidx $discarded_contigs
- awk 'BEGIN {FS="\\t"}; {print \$1 FS "0" FS \$2}' ${discarded_contigs}.fai > discarded_contigs.bed
-
-
- samtools view -@ ${task.cpus} -bL discarded_contigs.bed -U selected_contigs.bam $bam -o discarded_contigs.bam
-
-
- """
-}
-
-process EXTRACT_PAIRED_READS {
- tag "${meta.id}"
-
- input:
- tuple val(meta), path(bam)
-
- output:
- tuple val(meta), path("reads*.fastq.gz"), emit: reads
-
-
- script:
- """
-
- samtools fastq -@ ${task.cpus} -N -1 reads_R1.fastq.gz -2 reads_R2.fastq.gz $bam
-
- """
-}
-
-process EXTRACT_SINGLE_READS {
- tag "${meta.id}"
-
- input:
- tuple val(meta), path(bam)
-
- output:
- tuple val(meta), path("reads.fastq.gz"), emit: reads
-
- script:
- """
-
- samtools fastq -@ ${task.cpus} -o reads.fastq.gz $bam
-
- """
-}
-
-
-
-process MERGE_BAM_FILES {
- tag "${meta.id}"
-
- input:
- tuple val(meta), path(bam_discarded_reads), path(bam_selected_contigs)
-
- output:
- tuple val(meta), path("${meta.id}.filtered.sort.bam"), path("${meta.id}.filtered.sort.bam.bai") , emit: bam
-
- script:
-
- """
- samtools merge -@ ${task.cpus} $bam_discarded_reads $bam_selected_contigs -o merged.bam
-
- # reheader the bam to get only header corresponding to the final assembly only
- samtools view -H $bam_discarded_reads > filtered_assembly.header
- samtools reheader filtered_assembly.header merged.bam | samtools sort -@ ${task.cpus} -o ${meta.id}.filtered.sort.bam
-
- samtools index -@ ${task.cpus} ${meta.id}.filtered.sort.bam
-
- rm merged.bam
- """
-}
-
diff --git a/subworkflows/03_filtering.nf b/subworkflows/03_filtering.nf
index 3e7288b..72572c5 100644
--- a/subworkflows/03_filtering.nf
+++ b/subworkflows/03_filtering.nf
@@ -1,16 +1,26 @@
include { CHUNK_ASSEMBLY_FILTER; MERGE_ASSEMBLY_FILTER} from '../modules/filtering_cpm.nf'
include { QUAST } from '../modules/metaquast'
include { MINIMAP2; BWA_MEM } from '../modules/read_alignment'
-include { GET_ALIGNMENT_METRICS; FILTER_BAM; EXTRACT_SINGLE_READS; EXTRACT_PAIRED_READS; MERGE_BAM_FILES} from '../modules/read_alignment_manipulation'
+include { GET_ALIGNMENT_METRICS} from '../modules/read_alignment_manipulation'
workflow STEP_03_ASSEMBLY_FILTER {
take:
assemblies
+ reads
idxstats
bam
min_cpm
+
main:
+
+ filtering_outdir = "03_filtering/filtering_at_${params.min_contigs_cpm}cpm"
+
+ // if filtering has no effect on assembly. A symblink will be created between reads vs assembly of the step 02 in the step 03 outdir
+ unfiltered_assembly_bam_outdir = "02_assembly/02_3_reads_vs_primary_assembly"
+ filtered_assembly_bam_outdir = "${filtering_outdir}/03_2_reads_vs_filtered_assembly/"
+
+
ch_chunk_assembly_for_filter = assemblies
.splitFasta(by: 100000, file: true)
@@ -36,7 +46,8 @@ workflow STEP_03_ASSEMBLY_FILTER {
MERGE_ASSEMBLY_FILTER (
ch_grouped_selected,
ch_grouped_discarded,
- min_cpm
+ min_cpm,
+ "${filtering_outdir}/03_1_filtered_assembly"
)
ch_merged_selected = MERGE_ASSEMBLY_FILTER.out.merged_selected
discarded_contigs = MERGE_ASSEMBLY_FILTER.out.merged_discarded
@@ -49,51 +60,53 @@ workflow STEP_03_ASSEMBLY_FILTER {
with: !it[1].isEmpty()
}
+ samples_without_discarded_contigs = discarded_contigs_category.without.map{ it -> it[0]}
+ samples_with_discarded_contigs = discarded_contigs_category.with.map{ it -> it[0]}
- samples_without_discarded_contigs = discarded_contigs_category.without.map{ it -> it[0]}
- ch_bam_of_sample_without_discarded_contigs = samples_without_discarded_contigs.join(bam)
-
- ch_discarded_contigs_and_bam = discarded_contigs_category.with.join(bam)
- FILTER_BAM(ch_discarded_contigs_and_bam)
+ // make a symblink with the bam and bai from step 02 for samples that have not been affected by the filtering (no contig discarded)
+ result_path_dir = file("${params.outdir}/${filtered_assembly_bam_outdir}/")
+ result_path_dir.mkdirs()
+
+ samples_without_discarded_contigs.map {
+ sample -> { file("${result_path_dir}/${sample.id}/").mkdir()
+ file("${params.outdir}/${unfiltered_assembly_bam_outdir}/${sample.id}/${sample.id}.sort.bam")
+ .mklink("${result_path_dir}/${sample.id}/${sample.id}.sort.bam", overwrite:true)
+ file("${params.outdir}/${unfiltered_assembly_bam_outdir}/${sample.id}/${sample.id}.sort.bam.bai")
+ .mklink("${result_path_dir}/${sample.id}/${sample.id}.sort.bam.bai", overwrite:true)
+ }
+ }
- ch_discarded_bam = FILTER_BAM.out.discarded_contigs_bam
- ch_selected_bam = FILTER_BAM.out.selected_contigs_bam
+ ch_bam_unchanged_by_filtering = samples_without_discarded_contigs.join(bam)
- if ( params.type.toUpperCase() == "SR" ) {
-
- ch_discarded_reads = EXTRACT_PAIRED_READS(ch_discarded_bam)
+ ch_selected_contigs_and_reads = samples_with_discarded_contigs.join(ch_merged_selected).join(reads)
- ch_contigs_and_discarded_reads = ch_merged_selected.join(ch_discarded_reads)
- BWA_MEM(ch_contigs_and_discarded_reads, "03_filtering/03_2_reads_vs_filtered_assembly")
- ch_bam_with_discarded_reads = BWA_MEM.out.bam
- } else {
- ch_discarded_reads = EXTRACT_SINGLE_READS(ch_discarded_bam)
- ch_contigs_and_discarded_reads = ch_merged_selected.join(ch_discarded_reads)
- MINIMAP2(ch_contigs_and_discarded_reads, "03_filtering/03_2_reads_vs_filtered_assembly")
- ch_bam_with_discarded_reads = MINIMAP2.out.bam
-
+ if ( params.type.toUpperCase() == "SR" ) {
+ BWA_MEM(ch_selected_contigs_and_reads, filtered_assembly_bam_outdir)
+ ch_bam_post_filtering = BWA_MEM.out.bam
+ }
+ else {
+ MINIMAP2(ch_selected_contigs_and_reads, filtered_assembly_bam_outdir)
+ ch_bam_post_filtering = MINIMAP2.out.bam
}
- // remove bai from ch_bam_with_discarded_reads and add bam of selected contigs
- ch_bam_to_merge = ch_bam_with_discarded_reads.map{ it -> [it[0], it[1]]}.join(ch_selected_bam)
-
- ch_merged_bam = MERGE_BAM_FILES(ch_bam_to_merge)
-
- ch_final_bam = ch_bam_of_sample_without_discarded_contigs.mix(ch_merged_bam)
+ ch_all_bam = samples_without_discarded_contigs.join(bam).mix(ch_bam_post_filtering)
+ GET_ALIGNMENT_METRICS(ch_all_bam, filtered_assembly_bam_outdir)
- GET_ALIGNMENT_METRICS(ch_final_bam, '03_filtering/03_2_reads_vs_filtered_assembly/')
ch_flagstat = GET_ALIGNMENT_METRICS.out.sam_flagstat
ch_coverage = GET_ALIGNMENT_METRICS.out.sam_coverage
- QUAST( ch_merged_selected, "03_filtering/03_1_filtered_assembly/assembly_metric" )
+
+ QUAST( ch_merged_selected, "${filtering_outdir}/03_1_filtered_assembly/assembly_metric" )
ch_quast_report = QUAST.out.report
+ ch_final_bam = ch_bam_post_filtering.mix(ch_bam_unchanged_by_filtering)
+
emit:
selected_contigs = ch_merged_selected
quast_report = ch_quast_report
--
GitLab
From b28b6e765905460fbf2ef213bccb27134308b895 Mon Sep 17 00:00:00 2001
From: Jean Mainguy <jean.mainguy@inra.fr>
Date: Mon, 2 Jan 2023 11:53:50 +0100
Subject: [PATCH 166/194] add info on symb link of assembly
---
docs/output.md | 6 +++---
1 file changed, 3 insertions(+), 3 deletions(-)
diff --git a/docs/output.md b/docs/output.md
index f10e72a..ea47fa6 100644
--- a/docs/output.md
+++ b/docs/output.md
@@ -14,8 +14,8 @@ This document describes the output files produced by metagWGS.
- [**02\_assembly/02\_2\_deduplicated\_reads**](#02_assembly02_2_deduplicated_reads)
- [**02\_assembly/02\_3\_reads\_vs\_primary\_assembly**](#02_assembly02_3_reads_vs_primary_assembly)
- [**03\_filtering**](#03_filtering)
- - [**03\_filtering/03\_1\_filtered\_assembly/**](#03_filtering03_1_filtered_assembly)
- - [**03\_filtering/03\_2\_reads\_vs\_filtered\_assembly/**](#03_filtering03_2_reads_vs_filtered_assembly)
+ - [**03\_filtering/filtering\_at\_\<cpm\_threshold\>cpm/03\_1\_filtered\_assembly/**](#03_filteringfiltering_at_cpm_thresholdcpm03_1_filtered_assembly)
+ - [**03\_filtering/filtering\_at\_\<cpm\_threshold\>cpm/03\_2\_reads\_vs\_filtered\_assembly/**](#03_filteringfiltering_at_cpm_thresholdcpm03_2_reads_vs_filtered_assembly)
- [**04\_structural\_annot/**](#04_structural_annot)
- [**05\_protein\_alignment/05\_1\_database\_alignment/**](#05_protein_alignment05_1_database_alignment)
- [**06\_func\_annot**](#06_func_annot)
@@ -129,7 +129,7 @@ The `results/` directory contains a sub-directory for each step launched:
**NOTE:**
- This directory contains alignement metrics of reads aligned to the filtered assembly.
-- When the filtering step has no effect on the assembly (no contig are filtered out), the bam of the reads over the assembly does not change with the one from of this assembly tool `02_assembly/02_3_reads_vs_primary_assembly` adn consequently the bam found here will be a symbolic link with the bam from the assembly file.
+- When the filtering step has no effect on the assembly (no contig are filtered out), the bam of the reads over the assembly does not change with the one from the assembly step `02_assembly/02_3_reads_vs_primary_assembly` consequently the bam found here will be a symbolic link with the bam from the assembly file.
| File or directory/ | Description |
--
GitLab
From 6510a19ba75beee93728185358563dcbce9d39bb Mon Sep 17 00:00:00 2001
From: Jean Mainguy <jean.mainguy@inra.fr>
Date: Mon, 2 Jan 2023 11:58:27 +0100
Subject: [PATCH 167/194] remove prokka and replace with tools used
---
README.md | 2 +-
1 file changed, 1 insertion(+), 1 deletion(-)
diff --git a/README.md b/README.md
index 595160f..1300984 100644
--- a/README.md
+++ b/README.md
@@ -25,7 +25,7 @@ Many of these steps are optional and their necessity depends on the desired anal
* `S03_FILTERING`
* filters contigs with low CPM value ([Filter_contig_per_cpm.py](bin/Filter_contig_per_cpm.py) + [metaQUAST](http://quast.sourceforge.net/metaquast))
* `S04_STRUCTURAL_ANNOT`
- * makes a structural annotation of genes ([Prokka](https://github.com/tseemann/prokka) + [Rename_contigs_and_genes.py](bin/Rename_contigs_and_genes.py))
+ * makes a structural annotation of genes ([Prodigal](https://github.com/hyattpd/Prodigal) + [Barrnap](https://github.com/tseemann/barrnap) + [tRNAscan-SE](https://github.com/UCSC-LoweLab/tRNAscan-SE) [merge_annotations.py](bin/merge_annotations.py))
* `S05_ALIGNMENT`
* aligns reads to the contigs ([BWA-MEM2](https://github.com/bwa-mem2/bwa-mem2) or [Minimap2](https://github.com/lh3/minimap2) + [Samtools](http://www.htslib.org/))
* aligns the protein sequence of genes against a protein database ([DIAMOND](https://github.com/bbuchfink/diamond))
--
GitLab
From 725e3bd40c12e9ae0902cd09915a6e453f759bdd Mon Sep 17 00:00:00 2001
From: Jean Mainguy <jean.mainguy@inra.fr>
Date: Mon, 2 Jan 2023 12:00:04 +0100
Subject: [PATCH 168/194] fix typo
---
README.md | 2 +-
1 file changed, 1 insertion(+), 1 deletion(-)
diff --git a/README.md b/README.md
index 1300984..1a3736b 100644
--- a/README.md
+++ b/README.md
@@ -25,7 +25,7 @@ Many of these steps are optional and their necessity depends on the desired anal
* `S03_FILTERING`
* filters contigs with low CPM value ([Filter_contig_per_cpm.py](bin/Filter_contig_per_cpm.py) + [metaQUAST](http://quast.sourceforge.net/metaquast))
* `S04_STRUCTURAL_ANNOT`
- * makes a structural annotation of genes ([Prodigal](https://github.com/hyattpd/Prodigal) + [Barrnap](https://github.com/tseemann/barrnap) + [tRNAscan-SE](https://github.com/UCSC-LoweLab/tRNAscan-SE) [merge_annotations.py](bin/merge_annotations.py))
+ * makes a structural annotation of genes ([Prodigal](https://github.com/hyattpd/Prodigal) + [Barrnap](https://github.com/tseemann/barrnap) + [tRNAscan-SE](https://github.com/UCSC-LoweLab/tRNAscan-SE) + [merge_annotations.py](bin/merge_annotations.py))
* `S05_ALIGNMENT`
* aligns reads to the contigs ([BWA-MEM2](https://github.com/bwa-mem2/bwa-mem2) or [Minimap2](https://github.com/lh3/minimap2) + [Samtools](http://www.htslib.org/))
* aligns the protein sequence of genes against a protein database ([DIAMOND](https://github.com/bbuchfink/diamond))
--
GitLab
From 18fdd819ab3fdad8322cbda8089bc06a39e7b316 Mon Sep 17 00:00:00 2001
From: Vincent Darbot <vincent.darbot@inrae.fr>
Date: Fri, 6 Jan 2023 16:32:34 +0100
Subject: [PATCH 169/194] Change QUAST sample by sample to all samples at once
---
assets/multiqc_config.yaml | 4 ++--
main.nf | 6 ++----
modules/metaquast.nf | 16 +++++++---------
modules/multiqc.nf | 4 ++--
subworkflows/02_assembly.nf | 8 ++++++--
subworkflows/03_filtering.nf | 6 ++++--
6 files changed, 23 insertions(+), 21 deletions(-)
diff --git a/assets/multiqc_config.yaml b/assets/multiqc_config.yaml
index c24c305..40be5dc 100644
--- a/assets/multiqc_config.yaml
+++ b/assets/multiqc_config.yaml
@@ -52,7 +52,7 @@ module_order:
name: 'Quast primary assembly'
info: 'This section of the report shows primary assembly metrics.'
path_filters:
- - '*quast_primary/*/report.tsv'
+ - '*quast_primary/report.tsv'
- samtools:
name : 'Reads alignment on unfiltered assembly'
info: 'This section reports reads alignement on contigs.'
@@ -62,7 +62,7 @@ module_order:
name: 'Quast filtered assembly'
info: 'This section of the report shows metrics of the filtered assemblies.'
path_filters:
- - '*quast_filtered/*/report.tsv'
+ - '*quast_filtered/report.tsv'
- samtools:
name : 'Reads alignment on final assembly'
info: 'This section reports reads alignement on contigs.'
diff --git a/main.nf b/main.nf
index c46f90b..848dfb0 100644
--- a/main.nf
+++ b/main.nf
@@ -413,8 +413,7 @@ workflow {
ch_idxstats = S02_ASSEMBLY.out.idxstats
ch_unfilter_assembly_flagstat_report = S02_ASSEMBLY.out.flagstat
- ch_quast = S02_ASSEMBLY.out.assembly_report
- ch_quast_before_filter_report = ch_quast.map{ meta, quast_report -> quast_report }
+ ch_quast_before_filter_report = S02_ASSEMBLY.out.assembly_report
ch_software_versions_S02 = S02_ASSEMBLY.out.software_versions
}
@@ -439,8 +438,7 @@ workflow {
- ch_quast = S03_FILTERING.out.quast_report
- ch_quast_after_filter_report = ch_quast.map{ meta, quast_report -> quast_report }
+ ch_quast_after_filter_report = S03_FILTERING.out.quast_report
ch_sam_coverage = S03_FILTERING.out.sam_coverage
diff --git a/modules/metaquast.nf b/modules/metaquast.nf
index 9414136..bc20ad7 100644
--- a/modules/metaquast.nf
+++ b/modules/metaquast.nf
@@ -1,22 +1,20 @@
process QUAST {
- tag "${meta.id}"
label 'QUAST'
- publishDir "${params.outdir}/${outdir}/", mode: 'copy', pattern: "${meta.id}/*"
+ publishDir "${params.outdir}/${outdir}/", mode: 'copy'
input:
- tuple val(meta), path(assembly)
+ path(assemblies)
val outdir
output:
- path "${meta.id}/*", emit: all
- tuple val(meta), path ("${meta.id}/report.tsv"), emit: report
- path "v_quast.txt", emit: v_quast
+ path("assembly_metric/"), emit: all
+ path("assembly_metric/report.tsv"), emit: report
+ path("v_quast.txt"), emit: v_quast
script:
"""
- mkdir -p ${meta.id}/
- touch ${meta.id}/report.tsv
- metaquast.py --threads ${task.cpus} --rna-finding --max-ref-number 0 --min-contig 0 ${assembly} -o ${meta.id} --labels ${meta.id}
+ touch report.tsv
+ metaquast.py --threads ${task.cpus} --rna-finding --max-ref-number 0 --min-contig 0 ${assemblies} -o assembly_metric/
quast -v &> v_quast.txt
"""
diff --git a/modules/multiqc.nf b/modules/multiqc.nf
index 72c5951..c2a88c7 100644
--- a/modules/multiqc.nf
+++ b/modules/multiqc.nf
@@ -11,8 +11,8 @@ process MULTIQC {
path 'fastqc_clean_report/'
path 'kaiju_report/'
path "unfiltered_assembly_flagstat/"
- path 'quast_primary/*/report.tsv'
- path 'quast_filtered/*/report.tsv'
+ path 'quast_primary/report.tsv'
+ path 'quast_filtered/report.tsv'
path "final_assembly_flagstat/"
path 'prokka_report/'
path "featureCounts_report/"
diff --git a/subworkflows/02_assembly.nf b/subworkflows/02_assembly.nf
index d202dbd..d300ba9 100644
--- a/subworkflows/02_assembly.nf
+++ b/subworkflows/02_assembly.nf
@@ -48,7 +48,11 @@ workflow STEP_02_ASSEMBLY {
}
ch_assembly_renamed = RENAME_CONTIGS(ch_assembly)
- ASSEMBLY_QUAST( ch_assembly_renamed,"02_assembly/02_1_primary_assembly/assembly_metric/")
+ ch_assembly_quast = ch_assembly_renamed
+ .map { meta, file -> file }
+ .collect()
+
+ ASSEMBLY_QUAST( ch_assembly_quast,"02_assembly/02_1_primary_assembly/")
ch_assembly_report = ASSEMBLY_QUAST.out.report
ch_quast_v = ASSEMBLY_QUAST.out.v_quast
@@ -81,7 +85,7 @@ workflow STEP_02_ASSEMBLY {
ch_coverage = GET_ALIGNMENT_METRICS.out.sam_coverage
ch_samtools_v = GET_ALIGNMENT_METRICS.out.v_samtools
- ch_software_versions = ch_assembler_v.first().mix( ch_quast_v.first(),
+ ch_software_versions = ch_assembler_v.first().mix( ch_quast_v,
ch_bwa_mem_v.first(),
ch_minimap2_v.first(),
ch_samtools_v.first())
diff --git a/subworkflows/03_filtering.nf b/subworkflows/03_filtering.nf
index 72572c5..6ae3938 100644
--- a/subworkflows/03_filtering.nf
+++ b/subworkflows/03_filtering.nf
@@ -101,8 +101,10 @@ workflow STEP_03_ASSEMBLY_FILTER {
ch_flagstat = GET_ALIGNMENT_METRICS.out.sam_flagstat
ch_coverage = GET_ALIGNMENT_METRICS.out.sam_coverage
-
- QUAST( ch_merged_selected, "${filtering_outdir}/03_1_filtered_assembly/assembly_metric" )
+ ch_merged_quast = ch_merged_selected
+ .map { meta, file -> file }
+ .collect()
+ QUAST( ch_merged_quast, "${filtering_outdir}/03_1_filtered_assembly/" )
ch_quast_report = QUAST.out.report
ch_final_bam = ch_bam_post_filtering.mix(ch_bam_unchanged_by_filtering)
--
GitLab
From 9a4e843b73720380fe9486f782367f45b15a4f6f Mon Sep 17 00:00:00 2001
From: Vincent Darbot <vincent.darbot@inrae.fr>
Date: Mon, 9 Jan 2023 09:00:07 +0100
Subject: [PATCH 170/194] quantification_clusters.py: refactoring
---
bin/quantification_clusters.py | 289 +++++++++++++++++----------------
1 file changed, 153 insertions(+), 136 deletions(-)
diff --git a/bin/quantification_clusters.py b/bin/quantification_clusters.py
index 84c7367..b710902 100755
--- a/bin/quantification_clusters.py
+++ b/bin/quantification_clusters.py
@@ -30,154 +30,171 @@ __version__ = '0.1'
__email__ = 'support.bioinfo.genotoul@inra.fr'
__status__ = 'dev'
-# Status: dev.
-
# Modules importation.
import argparse
-import re
-import sys
+import logging
from datetime import datetime
+### Functions
+def parse_arguments():
+ '''
+ Parse parameters.
+ '''
+ parser = argparse.ArgumentParser(description = 'Script which create a \
+ correspondence table between global cluster id and gene id and \
+ a table with number of aligned reads in each sample and for each \
+ global cluster id.')
-# Print time.
-print(str(datetime.now()))
-
-# Manage parameters.
-parser = argparse.ArgumentParser(description = 'Script which create a \
-correspondence table between global cluster id and gene id and \
-a table with number of aligned reads in each sample and for each \
-global cluster id.')
+ parser.add_argument('-t', '--table_of_corespondences', required = True,
+ help = 'Correspondence table between global cluster \
+ id and intermediate cluster id.')
-parser.add_argument('-t', '--table_of_corespondences', required = True,
-help = 'Correspondence table between global cluster \
-id and intermediate cluster id.')
+ parser.add_argument('-l', '--list_of_file_clusters', required = True,
+ help = 'List of files containing correspondence tables between \
+ cluster intermediate cluster id and gene id per sample.')
-parser.add_argument('-l', '--list_of_file_clusters', required = True,
-help = 'List of files containing correspondence tables between \
-cluster intermediate cluster id and gene id per sample.')
+ parser.add_argument('-c', '--list_of_file_counts', required = True,
+ help = 'List of files storing read counts for each gene per sample.')
-parser.add_argument('-c', '--list_of_file_counts', required = True,
-help = 'List of files storing read counts for each gene per sample.')
+ parser.add_argument('-oc', '--output_counts', required = True,
+ help = 'Name of output file containing counts \
+ for each global cluster id and each sample.')
-parser.add_argument('-oc', '--output_counts', required = True,
-help = 'Name of output file containing counts \
-for each global cluster id and each sample.')
+ parser.add_argument('-oid', '--output_id', required = True,
+ help = 'Name of output file containing correspondence table \
+ between global cluster id and gene id.')
-parser.add_argument('-oid', '--output_id', required = True,
-help = 'Name of output file containing correspondence table \
-between global cluster id and gene id.')
+ parser.add_argument("--verbose", help="increase output verbosity",
+ action="store_true")
-parser.add_argument('-v', '--version', action = 'version', \
-version = __version__)
+ parser.add_argument('-v', '--version', action = 'version', \
+ version = __version__)
-args = parser.parse_args()
+ args = parser.parse_args()
+ return args
# Recovery of the list of file names.
-with open(args.list_of_file_counts) as fcounts_list:
- files_of_counts = fcounts_list.read().split()
-
-# For all variable names:
-# g_clstr: global cluster,
-# int_clstr: intermediate cluster,
-# gene: gene.
-
-# Dictionnaries d_g_clstr_id_by_int_clstr_id
-# and d_count_by_g_clstr initialization.
-d_g_clstr_id_by_int_clstr_id = {}
-d_count_by_g_clstr = {}
-
-with open(args.table_of_corespondences) as fp:
- for g_clstr_int_clstr_line in fp:
- g_clstr, *int_clstr = g_clstr_int_clstr_line.split()
- for clstr in int_clstr :
- d_g_clstr_id_by_int_clstr_id[clstr] = g_clstr
- d_count_by_g_clstr[g_clstr] = [0]*len(files_of_counts)
-
-print(d_g_clstr_id_by_int_clstr_id)
-print(d_count_by_g_clstr)
-
-
-# Initialization of dictionnary d_g_clstr_id_by_gene_id.
-d_g_clstr_id_by_gene_id = {}
-
-# Store into files_of_int_clstr_id_gene_id the list of sample files names
-# which contains correspondence between intermediate cluster id and gene id.
-with open(args.list_of_file_clusters) as fcluster_list:
- files_of_int_clstr_id_gene_id = fcluster_list.read().split()
-
-print(files_of_int_clstr_id_gene_id)
-
-# For each line of each sample file into files_of_int_clstr_id_gene_id,
-# store the gene id (key) in the dictionnary
-# d_g_clstr_id_by_gene_id.
-# The value of d_g_clstr_id_by_gene_id is the value of
-# d_g_clstr_id_by_int_clstr_id (global cluster id).
-
-for int_clstr_gene_path in files_of_int_clstr_id_gene_id:
- print(int_clstr_gene_path)
- with open(int_clstr_gene_path) as fh:
- for file_int_clstr_gene in fh:
- line_int_clstr_gene = file_int_clstr_gene.split()
- print(line_int_clstr_gene)
- int_clstr_id = line_int_clstr_gene[0]
- gene_id_from_clstr_gene_path = line_int_clstr_gene[1]
- if 'd_g_clstr_id_by_gene_id[gene_id_from_clstr_gene_path]' not in d_g_clstr_id_by_gene_id:
- d_g_clstr_id_by_gene_id[gene_id_from_clstr_gene_path] \
- = d_g_clstr_id_by_int_clstr_id[int_clstr_id]
- else:
- d_g_clstr_id_by_gene_id[gene_id_from_clstr_gene_path]\
- .append(d_g_clstr_id_by_int_clstr_id[int_clstr_id])
-
-print(d_g_clstr_id_by_gene_id)
-
-
-
-# For each count file (output of featureCounts), reading of lines one by one,
-# recovery of name of gene and count number and incrementing of corresponding
-# value in d_count_by_g_clstr.
-for (count_idx,counts_path) in enumerate(sorted(files_of_counts)):
- with open(counts_path) as fh:
- for f_gene_counts in fh:
- if f_gene_counts.startswith('#') \
- or f_gene_counts.startswith('Geneid'):
- continue
- line_gene_counts_split = f_gene_counts.split()
- gene_id = line_gene_counts_split[0].split("_gene")[0]
- gene_count = int(line_gene_counts_split[6])
- d_count_by_g_clstr[d_g_clstr_id_by_gene_id[gene_id]]\
- [count_idx] += gene_count
-
-
-
-#######################################
-# Write in the output files.
-#######################################
-
-# Write output file containing correspondence table
-# between global cluster id and gene id.
-with open(args.output_id,"w") as foutput_res_table:
- # Heading of output file: name of columns.
- foutput_res_table.write("seed_cluster" + "\t" + "id_gene" + "\n")
- # Writing seed cluster ids and genes ids for each sample contained in
- # d_g_clstr_id_by_gene_id in the output file line by line.
- for gene_id, g_clstr_id \
- in d_g_clstr_id_by_gene_id.items():
- foutput_res_table.write(g_clstr_id \
- + "\t" \
- + gene_id \
- + "\n")
-
-
-
-# Write output file containing global cluster id and read count for each sample.
-with open(args.output_counts,"w") as foutput_res_counts:
- # Heading of output file: name of columns.
- foutput_res_counts.write("seed_cluster\t" + "\t".join(files_of_counts) + "\n")
- # Writing global cluster ids and counts for each sample contained in
- # d_count_by_g_clstr in the output file line by line.
- for g_clstr, count in d_count_by_g_clstr.items():
- foutput_res_counts.write(g_clstr + "\t" \
- + "\t".join([str(i) for i in count])\
- + "\n")
-
+def processing_input_files(file_counts, correspondance_table, file_clusters):
+ '''
+ Recovering the list of file names.
+ For all variable names:
+ g_clstr: global cluster,
+ int_clstr: intermediate cluster,
+ gene: gene.
+
+ '''
+ with open(file_counts) as fcounts_list:
+ files_of_counts = fcounts_list.read().split()
+
+
+ d_g_clstr_id_by_int_clstr_id = {}
+ d_count_by_g_clstr = {}
+
+ with open(correspondance_table) as fp:
+ for g_clstr_int_clstr_line in fp:
+ g_clstr, *int_clstr = g_clstr_int_clstr_line.split()
+ for clstr in int_clstr :
+ d_g_clstr_id_by_int_clstr_id[clstr] = g_clstr
+ d_count_by_g_clstr[g_clstr] = [0]*len(files_of_counts)
+
+ d_g_clstr_id_by_gene_id = {}
+
+ # Store into files_of_int_clstr_id_gene_id the list of sample files names
+ # which contains correspondence between intermediate cluster id and gene id.
+ #
+ # For each line of each sample file into files_of_int_clstr_id_gene_id,
+ # store the gene id (key) in the dictionnary
+ # d_g_clstr_id_by_gene_id.
+ # The value of d_g_clstr_id_by_gene_id is the value of
+ # d_g_clstr_id_by_int_clstr_id (global cluster id).
+
+ with open(file_clusters) as fcluster_list:
+ files_of_int_clstr_id_gene_id = fcluster_list.read().split()
+
+ for int_clstr_gene_path in files_of_int_clstr_id_gene_id:
+ with open(int_clstr_gene_path) as fh:
+ for file_int_clstr_gene in fh:
+ line_int_clstr_gene = file_int_clstr_gene.split()
+ int_clstr_id = line_int_clstr_gene[0]
+ gene_id_from_clstr_gene_path = line_int_clstr_gene[1]
+ if 'd_g_clstr_id_by_gene_id[gene_id_from_clstr_gene_path]' not in d_g_clstr_id_by_gene_id:
+ d_g_clstr_id_by_gene_id[gene_id_from_clstr_gene_path] \
+ = d_g_clstr_id_by_int_clstr_id[int_clstr_id]
+ else:
+ d_g_clstr_id_by_gene_id[gene_id_from_clstr_gene_path]\
+ .append(d_g_clstr_id_by_int_clstr_id[int_clstr_id])
+
+ return files_of_counts, d_count_by_g_clstr, d_g_clstr_id_by_gene_id
+
+
+def linking_counts_and_clusters(files_of_counts, d_count_by_g_clstr, d_g_clstr_id_by_gene_id):
+ '''
+ For each count file (output of featureCounts), reading of lines one by one,
+ recovery of name of gene and count number and incrementing of corresponding
+ value in d_count_by_g_clstr.
+ '''
+ for (count_idx,counts_path) in enumerate(sorted(files_of_counts)):
+ with open(counts_path) as fh:
+ for f_gene_counts in fh:
+ if f_gene_counts.startswith('#') \
+ or f_gene_counts.startswith('Geneid'):
+ continue
+ line_gene_counts_split = f_gene_counts.split()
+ gene_id = line_gene_counts_split[0].split("_gene")[0]
+ gene_count = int(line_gene_counts_split[6])
+ d_count_by_g_clstr[d_g_clstr_id_by_gene_id[gene_id]]\
+ [count_idx] += gene_count
+
+ return d_count_by_g_clstr
+
+def writing_outputs( files_of_counts, d_count_by_g_clstr, d_g_clstr_id_by_gene_id, output_id, output_counts):
+ '''
+ # Write output file containing correspondence table
+ # between global cluster id and gene id.
+ '''
+ with open(output_id,"w") as foutput_res_table:
+ # Heading of output file: name of columns.
+ foutput_res_table.write("seed_cluster" + "\t" + "id_gene" + "\n")
+ # Writing seed cluster ids and genes ids for each sample contained in
+ # d_g_clstr_id_by_gene_id in the output file line by line.
+ for gene_id, g_clstr_id \
+ in d_g_clstr_id_by_gene_id.items():
+ foutput_res_table.write(g_clstr_id \
+ + "\t" \
+ + gene_id \
+ + "\n")
+
+ # Write output file containing global cluster id and read count for each sample.
+ with open(output_counts,"w") as foutput_res_counts:
+ # Heading of output file: name of columns.
+ foutput_res_counts.write("seed_cluster\t" + "\t".join(files_of_counts) + "\n")
+ # Writing global cluster ids and counts for each sample contained in
+ # d_count_by_g_clstr in the output file line by line.
+ for g_clstr, count in d_count_by_g_clstr.items():
+ foutput_res_counts.write(g_clstr + "\t" \
+ + "\t".join([str(i) for i in count])\
+ + "\n")
+
+def main():
+ args = parse_arguments()
+
+ if args.verbose:
+ logging.basicConfig(format="%(levelname)s: %(message)s", level=logging.DEBUG)
+ logging.info('Mode verbose ON')
+
+ else:
+ logging.basicConfig(format="%(levelname)s: %(message)s")
+
+ # Print time.
+ logging.info(str(datetime.now()))
+
+ files_of_counts, d_count_by_g_clstr, d_g_clstr_id_by_gene_id = \
+ processing_input_files(args.list_of_file_counts, args.table_of_corespondences, args.list_of_file_clusters)
+
+ d_count_by_g_clstr = linking_counts_and_clusters(files_of_counts, d_count_by_g_clstr, d_g_clstr_id_by_gene_id)
+
+ writing_outputs( files_of_counts, d_count_by_g_clstr, d_g_clstr_id_by_gene_id, args.output_id, args.output_counts)
+
+if __name__ == '__main__':
+ main()
--
GitLab
From 02ee802a5e45270da8f20c87dbf8ed2f28e60fc7 Mon Sep 17 00:00:00 2001
From: Vincent Darbot <vincent.darbot@inrae.fr>
Date: Mon, 9 Jan 2023 09:02:00 +0100
Subject: [PATCH 171/194] cd_hit_produce_table_clstr.py: add fasta.clstr file
as input and change the script's call in cd-hit.nf
---
bin/cd_hit_produce_table_clstr.py | 77 ++++++++++++++++++++-----------
modules/cd_hit.nf | 3 +-
2 files changed, 51 insertions(+), 29 deletions(-)
diff --git a/bin/cd_hit_produce_table_clstr.py b/bin/cd_hit_produce_table_clstr.py
index 1e44d50..5fafa9b 100755
--- a/bin/cd_hit_produce_table_clstr.py
+++ b/bin/cd_hit_produce_table_clstr.py
@@ -1,30 +1,53 @@
#!/usr/bin/env python
import sys, re
+from argparse import ArgumentParser, ArgumentDefaultsHelpFormatter
-#init dictionaries:
-ref = ""
-seqs = []
-
-while 1 :
- line = sys.stdin.readline()
- #print line
- if line == '' :
- break
- else :
- if line[0] == ">":
- for seq in seqs :
- print(ref+"\t"+seq)
- ref = ""
- seqs = []
- else:
- a, b = line.split('>', 1)
- name = b.split("...")[0]
- rep = (line.rstrip()[-1] == '*')
- if rep :
- ref = name
- seqs.append(name)
- else :
- seqs.append(name)
-
-for seq in seqs :
- print(ref+"\t"+seq)
+def parse_arguments():
+ """Parse script arguments."""
+ parser = ArgumentParser(description="...",
+ formatter_class=ArgumentDefaultsHelpFormatter)
+ parser.add_argument('-i', '--input_file', required=True, help="cd-hit output file of representating clusters.")
+
+ parser.add_argument('-o', '--output_file', required=True, help="Clusters table.")
+
+ args = parser.parse_args()
+ return args
+
+def process(input_file, output_file):
+ #init dictionaries:
+ ref = ""
+ seqs = []
+
+ FH_out = open(output_file, 'wt')
+ FH_input = open(input_file)
+ for line in FH_input:
+ #print line
+ if line == '' :
+ break
+ else :
+ if line[0] == ">":
+ for seq in seqs :
+ FH_out.write(ref+"\t"+seq+"\n")
+ ref = ""
+ seqs = []
+ else:
+ a, b = line.split('>', 1)
+ name = b.split("...")[0]
+ rep = (line.rstrip()[-1] == '*')
+ if rep :
+ ref = name
+ seqs.append(name)
+ else :
+ seqs.append(name)
+
+ for seq in seqs :
+ FH_out.write(ref+"\t"+seq+"\n")
+
+def main():
+ args = parse_arguments()
+
+ process(args.input_file, args.output_file)
+
+
+if __name__ == '__main__':
+ main()
\ No newline at end of file
diff --git a/modules/cd_hit.nf b/modules/cd_hit.nf
index 217bc92..3896ec1 100644
--- a/modules/cd_hit.nf
+++ b/modules/cd_hit.nf
@@ -35,13 +35,12 @@ process GLOBAL_CD_HIT {
path "All-cd-hit-est.${pct_id}.fasta", emit: fasta_clusters
path "table_clstr.txt", emit: clstr_table
-
script:
"""
# *fasta is important to get the correct order
cat *.fasta > All-cd-hit-est.${pct_id}
cd-hit-est -c ${pct_id} -i All-cd-hit-est.${pct_id} -o All-cd-hit-est.${pct_id}.fasta -T ${task.cpus} -M ${task.mem} -d 150
- cat All-cd-hit-est.${pct_id}.fasta.clstr | cd_hit_produce_table_clstr.py > table_clstr.txt
+ cd_hit_produce_table_clstr.py -i All-cd-hit-est.${pct_id}.fasta.clstr -o table_clstr.txt
"""
}
--
GitLab
From e7c49fc1a457f701b6dddc38b9cbb6cf0814ed15 Mon Sep 17 00:00:00 2001
From: Vincent Darbot <vincent.darbot@inrae.fr>
Date: Mon, 9 Jan 2023 09:03:04 +0100
Subject: [PATCH 172/194] merge_contig_quantif_perlineage.py create
generate_krona_directories() refactoring
---
bin/merge_contig_quantif_perlineage.py | 25 ++++++++++++-------------
bin/merge_kaiju_results.py | 2 --
2 files changed, 12 insertions(+), 15 deletions(-)
diff --git a/bin/merge_contig_quantif_perlineage.py b/bin/merge_contig_quantif_perlineage.py
index 8dd82ec..da9cc06 100755
--- a/bin/merge_contig_quantif_perlineage.py
+++ b/bin/merge_contig_quantif_perlineage.py
@@ -48,6 +48,15 @@ def parse_arguments():
args = parser.parse_args()
return args
+def generate_krona_directories(path, paramater,name, df_reads_count, ranks_lineage):
+ '''
+ path: Directory output name
+ paramater: paramater analyzed (either nb_reads or depth
+ '''
+ read_abd_dir = path
+ os.makedirs(read_abd_dir, exist_ok=True)
+ outfile = os.path.join(read_abd_dir, f'{name}.krona')
+ df_reads_count[[paramater ] + ranks_lineage].to_csv(outfile, index=False, header=False, sep="\t")
def main():
@@ -131,19 +140,9 @@ def main():
df_reads_count = df_reads_count.replace(' None', "")
# number of reads abundance
- read_abd_dir = "krona_reads_count_abundance"
- os.makedirs(read_abd_dir, exist_ok=True)
- outfile = os.path.join(read_abd_dir, f'{name}.krona')
- df_reads_count[["nb_reads" ] + ranks_lineage].to_csv(outfile, index=False, header=False, sep="\t")
-
- # mean depth abundance
- depth_abd_dir = "krona_mean_depth_abundance"
- os.makedirs(depth_abd_dir, exist_ok=True)
- outfile = os.path.join(depth_abd_dir, f'{name}.krona')
- df_reads_count[["depth" ] + ranks_lineage].to_csv(outfile, index=False, header=False, sep="\t")
-
-
-
+ generate_krona_directories("krona_reads_count_abundance", "nb_reads", name, df_reads_count, ranks_lineage)
+ # mean depth abundance
+ generate_krona_directories("krona_mean_depth_abundance", "depth", name, df_reads_count, ranks_lineage)
if __name__ == '__main__':
main()
diff --git a/bin/merge_kaiju_results.py b/bin/merge_kaiju_results.py
index 25cdb1c..1c8994d 100755
--- a/bin/merge_kaiju_results.py
+++ b/bin/merge_kaiju_results.py
@@ -18,8 +18,6 @@ __version__ = '0.1'
__email__ = 'support.bioinfo.genotoul@inra.fr'
__status__ = 'dev'
-# Status: dev.
-
# Modules importation.
import argparse
--
GitLab
From 470e367584ba905dcbd9b8d5b89bdcb2eb12e8b2 Mon Sep 17 00:00:00 2001
From: VIENNE MAINA <maina.vienne@inrae.fr>
Date: Tue, 10 Jan 2023 16:15:19 +0100
Subject: [PATCH 173/194] Add coassembly
---
conf/functional_test.config | 2 +-
docs/usage.md | 5 +-
main.nf | 62 ++---------
modules/assembly.nf | 26 +++--
modules/binning.nf | 4 +-
modules/cd_hit.nf | 3 +-
modules/feature_counts.nf | 6 +-
modules/read_alignment.nf | 16 +--
nextflow.config | 2 +
subworkflows/02_assembly.nf | 156 +++++++++++++++++++++------
subworkflows/03_filtering.nf | 143 +++++++++++++++---------
subworkflows/04_structural_annot.nf | 2 -
subworkflows/05_protein_alignment.nf | 2 +-
subworkflows/06_functionnal_annot.nf | 17 ++-
subworkflows/07_taxonomic_affi.nf | 15 ++-
subworkflows/08_binning.nf | 72 +++++++++----
16 files changed, 345 insertions(+), 188 deletions(-)
diff --git a/conf/functional_test.config b/conf/functional_test.config
index b1fa154..6268237 100644
--- a/conf/functional_test.config
+++ b/conf/functional_test.config
@@ -5,7 +5,7 @@ singularity.enabled = true
singularity.autoMounts = true
process {
- container = "/work/project/plateforme/metaG/functional_test/singularity_img/metagwgs.sif"
+ container = "/work/project/plateforme/metaG/functional_test/singularity_img/dev_v2.4/metagwgs.sif"
withLabel: BINNING {
container = "/work/project/plateforme/metaG/functional_test/singularity_img/binning.sif"
}
diff --git a/docs/usage.md b/docs/usage.md
index ed48bf6..a0907e2 100644
--- a/docs/usage.md
+++ b/docs/usage.md
@@ -118,7 +118,7 @@ For HiFi the fastq2 column is not needed
> name_sample2,number_flowcell,path_sample2_R1.fastq.gz,path_sample2_R2.fastq.gz
> ```
- * **If you want to perfom cross alignment for binning on groups** :
+ * **If you want to perfom coassembly or cross alignment for binning on groups** :
> ```
> sample,group,fastq_1,fastq_2
> name_sample1,group_name,path_sample1_flowcell1_R1.fastq.gz,path_sample1_flowcell1_R2.fastq.gz
@@ -276,6 +276,7 @@ No parameter available for this substep.
* `--assembly` allows to indicate the assembly tool. For short reads: `["metaspades" or "megahit"]`: Default: `metaspades`.
For HiFi reads: `["hifiasm-meta", "metaflye"]`. Default: `hifiasm-meta`.
+* `--coassembly` allows to assemble together the samples labeled with the same group in the samplesheet. It will generate one assembly for each group. To co-assemble all of your samples together, you must indicate a unique group for each sample in the samplesheet. **WARNING** With the coassembly, you can't use `--binning_cross_alignment 'group'` because one binning will be generate for each group co-assembled and automatically mapping with every sample of his group but `--binning_cross_alignment 'all'` can be use to cross align every sample with every group.
**WARNING 4:** For short reads, the user can choose between `metaspades` or `megahit` for `--assembly` parameter. The choice can be based on CPUs and memory availability: `metaspades` needs more CPUs and memory than `megahit` but our tests showed that assembly metrics are better for `metaspades` than `megahit`.For PacBio HiFi reads, the user can choose between `hifiasm-meta` or `metaflye`.
@@ -352,6 +353,8 @@ For example check the version of software in the yaml file and the singularity r
* `--gtdbtk_bank`: indicates path to the GTDBTK database.
+* `--metabat2_seed`: Set the seed for metabat2, for exact reproducibility of metabat2 (default: 0 (random seed))
+
* `--binning_cross_alignment ["all","group","individual"]`: defines mapping strategy to compute co-abundances for binning. `all` means that each samples will be mapped against every assembly, `group` means that all sample from a group will be mapped against every assembly of the group, `individual` means that each sample will only be mapped against his assembly. Default `individual`
diff --git a/main.nf b/main.nf
index 848dfb0..ac14614 100644
--- a/main.nf
+++ b/main.nf
@@ -23,7 +23,7 @@ include { STEP_08_BINNING as S08_BINNING } from './subworkflows/08_binning'
include { GET_SOFTWARE_VERSIONS } from './modules/get_software_versions'
include { MULTIQC } from './modules/multiqc'
-include { MERGE_FASTQ } from './modules/merge_fastq.nf'
+
/*
@@ -65,6 +65,7 @@ include { MERGE_FASTQ } from './modules/merge_fastq.nf'
--stop_at_assembly Stop the pipeline at this step
--assembly Indicate the assembly tool for short reads ["metaspades" or "megahit" ]. Default: "metaspades".
or for HiFi reads ["hifiasm-meta", "metaflye"]. Default: "hifiasm-meta".
+ --coassembly Assemble together samples labeled with the same group in the samplesheet
S03_FILTERING options:
--stop_at_filtering Stop the pipeline at this step
@@ -213,6 +214,10 @@ workflow {
exit 1, "You must specify --skip_binning or specify a GTDB-TK bank with --gtdbtk_bank"
}
+ if ( params.coassembly && params.binning_cross_alignment == 'group'){
+ exit 1, "--binning_cross_alignment group must not be use use --coassembly."
+ }
+
////////////
@@ -228,8 +233,8 @@ workflow {
if (hasExtension(row.fastq_1, "fastq") || hasExtension(row.fastq_1, "fq") || hasExtension(row.fastq_2, "fastq") || hasExtension(row.fastq_2, "fq")) {
exit 1, "We do recommend to use gziped fastq file to help you reduce your data footprint."
}
- if (params.binning_cross_alignment == 'group' && row.group == null){
- exit 1, "You must specify groups in the samplesheet if you want to use --binning_cross_alignment 'group'"
+ if ((params.binning_cross_alignment == 'group' || params.coassembly) && row.group == null){
+ exit 1, "You must specify groups in the samplesheet if you want to use --binning_cross_alignment 'group' or --coassembly"
}
["sample":row.sample,
"flowcell":row.flowcell,
@@ -244,7 +249,6 @@ workflow {
def meta = [:]
meta.id = item.sample
if (item.flowcell!=null) { meta.id = meta.id+"_"+item.flowcell}
- if (item.group !=null) {meta.id = meta.id+"_"+item.group}
meta.sample = item.sample
meta.flowcell = item.flowcell
meta.group = item.group
@@ -265,7 +269,6 @@ workflow {
def meta = [:]
meta.id = item.sample
if (item.flowcell!=null) { meta.id = meta.id+"_"+item.flowcell}
- if (item.group !=null) {meta.id = meta.id+"_"+item.group}
meta.sample = item.sample
meta.flowcell = item.flowcell
meta.group = item.group
@@ -346,7 +349,7 @@ workflow {
ch_host_index,
ch_kaiju_db
)
- ch_preprocessed_reads = S01_CLEAN_QC.out.preprocessed_reads
+ ch_reads = S01_CLEAN_QC.out.preprocessed_reads
ch_cutadapt_report = S01_CLEAN_QC.out.cutadapt_report
ch_sickle_report = S01_CLEAN_QC.out.sickle_report
@@ -357,54 +360,11 @@ workflow {
ch_kaiju_report = S01_CLEAN_QC.out.kaiju_report
ch_software_versions_S01 = S01_CLEAN_QC.out.software_versions
- } else {
- ch_preprocessed_reads = ch_reads
}
if ( !params.stop_at_clean ) {
- if (!has_assembly & has_flowcell ){
- //////////////////
- // Manage Flowcell
- //////////////////
- ch_reads_tmp = ch_preprocessed_reads
- .map {
- meta, fastq ->
- [ meta.sample, meta, fastq ]
- }
- .groupTuple(by: [0])
- .branch {
- id, meta, fastq ->
- single : fastq.size() == 1
- return [[id:meta.sample.unique().join(),
- sample:meta.sample.unique().join(),
- flowcell:meta.flowcell.join("_"),
- group:meta.group.unique().join(),
- assembly:meta.assembly.unique().join(),
- type:meta.type.unique().join()], fastq.flatten() ]
- multiple: fastq.size() > 1
- return [[id:meta.sample.unique().join(),
- sample:meta.sample.unique().join(),
- flowcell:meta.flowcell.join("_"),
- group:meta.group.unique().join(),
- assembly:meta.assembly.unique().join(),
- type:meta.type.unique().join()], fastq.flatten() ]
- }
-
-
- MERGE_FASTQ (
- ch_reads_tmp.multiple
- )
- .reads
- .mix(ch_reads_tmp.single)
- .set{ch_preprocessed_reads}
- }
-
- /////////////////////
- //End manage Flowcell
- /////////////////////
-
- S02_ASSEMBLY ( ch_preprocessed_reads, ch_assembly, has_assembly, assembly_tool )
+ S02_ASSEMBLY ( ch_reads, ch_assembly, has_assembly, assembly_tool, has_flowcell )
ch_assembly = S02_ASSEMBLY.out.assembly
ch_reads = S02_ASSEMBLY.out.reads
ch_bam = S02_ASSEMBLY.out.bam
@@ -426,7 +386,7 @@ workflow {
S03_FILTERING (
ch_assembly,
- ch_preprocessed_reads,
+ ch_reads,
ch_idxstats,
ch_bam,
ch_min_contigs_cpm,
diff --git a/modules/assembly.nf b/modules/assembly.nf
index 98cf3c6..5c24b43 100644
--- a/modules/assembly.nf
+++ b/modules/assembly.nf
@@ -4,7 +4,7 @@ process METASPADES {
label 'ASSEMBLY_SR'
input:
- tuple val(meta), path(reads)
+ tuple val(meta), path(read1), path(read2)
output:
tuple val(meta), path("metaspades_${meta.id}/${meta.id}.contigs.fa"), emit: assembly
@@ -15,7 +15,21 @@ process METASPADES {
(_,mem,unit) = (task.memory =~ /(\d+) ([A-Z]B)/)[0]
if ( unit =~ /GB/ ) {
"""
- metaspades.py -t ${task.cpus} -m $mem -1 ${reads[0]} -2 ${reads[1]} -o metaspades_${meta.id}
+ echo "
+[
+ {
+ orientation: \\"fr\\",
+ type: \\"paired-end\\",
+ right reads: [
+ \\"${read1.join('\\",\n \\"')}\\"
+ ],
+ left reads: [
+ \\"${read2.join('\\",\n \\"')}\\"
+ ]
+ }
+]" > input.yaml
+
+ metaspades.py -t ${task.cpus} -m $mem --dataset input.yaml -o metaspades_${meta.id}
mv metaspades_${meta.id}/scaffolds.fasta metaspades_${meta.id}/${meta.id}.contigs.fa
mv metaspades_${meta.id}/spades.log metaspades_${meta.id}/${meta.id}.log
mv metaspades_${meta.id}/params.txt metaspades_${meta.id}/${meta.id}.params.txt
@@ -34,7 +48,7 @@ process MEGAHIT {
label 'ASSEMBLY_SR'
input:
- tuple val(meta), path(reads)
+ tuple val(meta), path(read1), path(read2)
output:
tuple val(meta), path("megahit_${meta.id}/${meta.id}.contigs.fa"), emit: assembly
@@ -43,7 +57,7 @@ process MEGAHIT {
script:
"""
- megahit -t ${task.cpus} -1 ${reads[0]} -2 ${reads[1]} -o megahit_${meta.id} --out-prefix "${meta.id}"
+ megahit -t ${task.cpus} -1 ${read1.join(',')} -2 ${read2.join(',')} -o megahit_${meta.id} --out-prefix "${meta.id}"
mv megahit_${meta.id}/options.json megahit_${meta.id}/${meta.id}.params.txt
rm -r megahit_${meta.id}/intermediate_contigs
@@ -69,7 +83,7 @@ process HIFIASM_META {
"""
mkdir hifiasm_meta_${meta.id}
- hifiasm_meta -t ${task.cpus} -o ${meta.id} $reads 2> hifiasm_meta_${meta.id}/${meta.id}.log
+ hifiasm_meta -t ${task.cpus} -o ${meta.id} ${reads.join(' ')} 2> hifiasm_meta_${meta.id}/${meta.id}.log
# gfa to fasta format
awk '/^S/{print ">"\$2"\\n"\$3}' ${meta.id}.p_ctg.gfa | fold > hifiasm_meta_${meta.id}/${meta.id}.contigs.fa
@@ -98,7 +112,7 @@ process METAFLYE {
"""
mkdir metaflye_${meta.id}
- flye --pacbio-hifi $reads -o 'metaflye_${meta.id}' --meta -t ${task.cpus} 2> metaflye_${meta.id}/${meta.id}.log
+ flye --pacbio-hifi ${reads.join(' ')} -o 'metaflye_${meta.id}' --meta -t ${task.cpus} 2> metaflye_${meta.id}/${meta.id}.log
mv metaflye_${meta.id}/assembly.fasta metaflye_${meta.id}/${meta.id}.contigs.fa
mv metaflye_${meta.id}/params.json metaflye_${meta.id}/${meta.id}.params.json
diff --git a/modules/binning.nf b/modules/binning.nf
index c90306a..431fb01 100644
--- a/modules/binning.nf
+++ b/modules/binning.nf
@@ -30,7 +30,7 @@ process METABAT2 {
script:
"""
mkdir -p metabat2/bins/
- metabat2 --inFile $fna --abdFile $depth --outFile metabat2/bins/${meta.id}_metabat2 --numThreads ${task.cpus}
+ metabat2 --inFile $fna --abdFile $depth --outFile metabat2/bins/${meta.id}_metabat2 --numThreads ${task.cpus} --seed ${params.metabat2_seed}
echo \$(metabat2 -h 2>&1) > v_metabat2.txt
"""
}
@@ -130,7 +130,7 @@ process METAWRAP_REFINMENT {
path "v_metawrap.txt", emit: v_metawrap
script:
- bins_flag = (bins3 != null) ? "-A $bins1 -B $bins2 -C $bins3" : "-A $bins2 -B $bins2 "
+ bins_flag = (bins3 != null) ? "-A $bins1 -B $bins2 -C $bins3" : "-A $bins1 -B $bins2 "
"""
echo "metawrap 1.3_modified" > v_metawrap.txt
diff --git a/modules/cd_hit.nf b/modules/cd_hit.nf
index 3896ec1..b341142 100644
--- a/modules/cd_hit.nf
+++ b/modules/cd_hit.nf
@@ -15,7 +15,7 @@ process INDIVIDUAL_CD_HIT {
script:
"""
cd-hit-est -c ${pct_id} -i ${ffn} -o ${meta.id}.cd-hit-est.${pct_id}.fasta -T ${task.cpus} -M ${task.mem} -d 150
- cat ${meta.id}.cd-hit-est.${pct_id}.fasta.clstr | cd_hit_produce_table_clstr.py > ${meta.id}.cd-hit-est.${pct_id}.table_cluster_contigs.txt
+ cd_hit_produce_table_clstr.py -i ${meta.id}.cd-hit-est.${pct_id}.fasta.clstr -o ${meta.id}.cd-hit-est.${pct_id}.table_cluster_contigs.txt
echo \$(cd-hit -h 2>&1) > v_cdhit.txt
"""
}
@@ -54,7 +54,6 @@ main:
INDIVIDUAL_CD_HIT( ch_assembly, ch_percentage_identity )
ch_individual_clusters = INDIVIDUAL_CD_HIT.out.clstr_fasta.collect()
GLOBAL_CD_HIT(ch_individual_clusters , ch_percentage_identity )
- ch_ffn = ch_assembly.flatMap{it -> it[1]}.collect()
emit:
individual_clstr_table = INDIVIDUAL_CD_HIT.out.clstr_table
diff --git a/modules/feature_counts.nf b/modules/feature_counts.nf
index 73ec38c..63eb79a 100644
--- a/modules/feature_counts.nf
+++ b/modules/feature_counts.nf
@@ -44,14 +44,12 @@ process QUANTIFICATION_TABLE {
workflow QUANTIFICATION {
take:
- ch_gff // channel: [ val(meta), path(gff) ]
- ch_bam // channel: [ val(meta), path(bam), path(bam_index) ]
+ ch_gff_and_bam // channel: [ val(meta), path(gff), path(bam), path(bam_index) ]
ch_individual_clstr_table
ch_global_clstr_table
main:
- ch_gff_and_bam = ch_gff.join(ch_bam, remainder: false)
-
+
FEATURE_COUNTS(ch_gff_and_bam)
ch_count_table = FEATURE_COUNTS.out.count_table.collect()
ch_quant_report = FEATURE_COUNTS.out.summary
diff --git a/modules/read_alignment.nf b/modules/read_alignment.nf
index 881f6c8..5183c8b 100644
--- a/modules/read_alignment.nf
+++ b/modules/read_alignment.nf
@@ -1,6 +1,6 @@
process BWA_MEM {
tag "${meta.id}"
- publishDir "${params.outdir}/$publishDir_path/${meta.id}", mode: 'copy', pattern:"*sort*"
+ publishDir "${params.outdir}/$publishDir_path/${meta.id}", mode: 'copy', pattern:"*bam*"
input:
tuple val(meta), path(fna), path(reads)
@@ -8,16 +8,16 @@ process BWA_MEM {
output:
- tuple val(meta), path("${meta.id}.sort.bam"), path("${meta.id}.sort.bam.bai"), emit: bam
+ tuple val(meta), path("${meta.id}.bam"), path("${meta.id}.bam.bai"), emit: bam
path "v_bwa.txt", emit: v_bwa_mem2
path "v_samtools.txt", emit: v_samtools
script:
"""
bwa-mem2 index ${fna} -p ${fna}
- bwa-mem2 mem -t ${task.cpus} ${fna} ${reads[0]} ${reads[1]} | samtools view -@ ${task.cpus} -bS - | samtools sort -@ ${task.cpus} - -o ${meta.id}.sort.bam
+ bwa-mem2 mem -t ${task.cpus} ${fna} ${reads[0]} ${reads[1]} | samtools view -@ ${task.cpus} -bS - | samtools sort -@ ${task.cpus} - -o ${meta.id}.bam
- samtools index -@ ${task.cpus} ${meta.id}.sort.bam
+ samtools index -@ ${task.cpus} ${meta.id}.bam
bwa-mem2 version > v_bwa.txt
samtools --version &> v_samtools.txt
@@ -27,23 +27,23 @@ process BWA_MEM {
process MINIMAP2 {
tag "${meta.id}"
label 'MINIMAP2'
- publishDir "${params.outdir}/$publishDir_path/${meta.id}", mode: 'copy', pattern:"*sort*"
+ publishDir "${params.outdir}/$publishDir_path/${meta.id}", mode: 'copy', pattern:"*bam*"
input:
tuple val(meta), path(fna), path(reads)
val(publishDir_path)
output:
- tuple val(meta), path("${meta.id}.sort.bam"), path("${meta.id}.sort.bam.bai"), emit: bam
+ tuple val(meta), path("${meta.id}.bam"), path("${meta.id}.bam.bai"), emit: bam
path "v_minimap2.txt", emit: v_minimap2
path "v_samtools.txt", emit: v_samtools
script:
"""
# align reads to contigs, keep only primary aln and sort resulting bam
- minimap2 -t ${task.cpus} -ax map-hifi $fna $reads | samtools view -@ ${task.cpus} -b -F 2304 | samtools sort -@ ${task.cpus} -o ${meta.id}.sort.bam
+ minimap2 -t ${task.cpus} -ax map-hifi $fna $reads | samtools view -@ ${task.cpus} -b -F 2304 | samtools sort -@ ${task.cpus} -o ${meta.id}.bam
- samtools index ${meta.id}.sort.bam -@ ${task.cpus}
+ samtools index ${meta.id}.bam -@ ${task.cpus}
samtools --version &> v_samtools.txt
minimap2 --version &> v_minimap2.txt
diff --git a/nextflow.config b/nextflow.config
index 1516f4c..b0f4b71 100644
--- a/nextflow.config
+++ b/nextflow.config
@@ -21,6 +21,7 @@ params {
gtdbtk_bank = ""
percentage_identity = 0.95
type = ""
+ coassembly = false
// Stop after step or skip optional step/sub-step.
@@ -58,6 +59,7 @@ params {
drep_threshold = 0.95
min_completeness = 50
max_contamination = 10
+ metabat2_seed = 0
// Ressources.
kaiju_db_dir = false
diff --git a/subworkflows/02_assembly.nf b/subworkflows/02_assembly.nf
index d300ba9..46c4da5 100644
--- a/subworkflows/02_assembly.nf
+++ b/subworkflows/02_assembly.nf
@@ -3,13 +3,15 @@ include { QUAST as ASSEMBLY_QUAST} from '../modules/metaquast'
include { READS_DEDUPLICATION } from '../modules/reads_deduplication'
include { MINIMAP2 } from '../modules/read_alignment'
include { GET_ALIGNMENT_METRICS } from '../modules/read_alignment_manipulation'
+include { MERGE_FASTQ } from '../modules/merge_fastq.nf'
workflow STEP_02_ASSEMBLY {
take:
- reads
- assembly
- has_assembly
- assembly_tool
+ reads
+ assembly
+ has_assembly
+ assembly_tool
+ has_flowcell
main:
ch_assembler_v = Channel.empty()
@@ -17,36 +19,107 @@ workflow STEP_02_ASSEMBLY {
ch_bwa_mem_v = Channel.empty()
ch_minimap2_v = Channel.empty()
ch_samtools_v = Channel.empty()
-
- if (has_assembly){
- ch_assembly = assembly
- }
- else {
- if(assembly_tool == 'metaspades') {
- METASPADES(reads)
- ch_assembly = METASPADES.out.assembly
- ch_assembler_v = METASPADES.out.v_metaspades
- }
- else if(assembly_tool == 'megahit') {
- MEGAHIT(reads)
- ch_assembly = MEGAHIT.out.assembly
- ch_assembler_v = MEGAHIT.out.v_megahit
- }
- else if(assembly_tool == 'hifiasm-meta') {
- HIFIASM_META(reads)
- ch_assembly = HIFIASM_META.out.assembly
- ch_assembler_v = HIFIASM_META.out.v_hifiasm_meta
- }
- else if(assembly_tool == 'metaflye') {
- METAFLYE(reads)
+
+
+ ch_assembly = assembly
+ ch_reads = reads
+
+ if (!has_assembly & has_flowcell ){
+ //////////////////
+ // Manage Flowcell
+ //////////////////
+ ch_reads_flowcell = reads
+ .map { meta, fastq ->
+ [ meta.sample, meta, fastq ] }
+ .groupTuple(by: [0])
+ .branch { id, meta, fastq ->
+ single : fastq.size() == 1
+ return [[id:meta.sample.unique().join(),
+ sample:meta.sample.unique().join(),
+ flowcell:meta.flowcell.join("_"),
+ group:meta.group.unique().join(),
+ assembly:meta.assembly.unique().join(),
+ type:meta.type.unique().join()], fastq.flatten() ]
+ multiple: fastq.size() > 1
+ return [[id:meta.sample.unique().join(),
+ sample:meta.sample.unique().join(),
+ flowcell:meta.flowcell.join("_"),
+ group:meta.group.unique().join(),
+ assembly:meta.assembly.unique().join(),
+ type:meta.type.unique().join()], fastq.flatten() ]
+ }
+
+ MERGE_FASTQ (ch_reads_flowcell.multiple)
+ .reads
+ .mix(ch_reads_flowcell.single)
+ .set{ch_reads}
+ }
+
+ if (params.coassembly){
+ ch_reads.map { meta, fastq ->
+ [ meta.group, meta, fastq] }
+ .groupTuple(by: [0])
+ .map { group, metas, fastq ->
+ def meta = [:]
+ meta.id = metas.group.unique().join()
+ meta.sample = metas.sample.join("_")
+ meta.flowcell = metas.flowcell.unique().join()
+ meta.group = metas.group.unique().join()
+ meta.assembly = metas.assembly.unique().join()
+ meta.type = metas.type.unique().join()
+ if (params.type.toUpperCase() == "SR") {
+ return [meta, fastq.collect { it[0] }, fastq.collect { it[1] }]
+ } else {
+ return [meta, fastq ]
+ }}
+ .set { ch_reads_assembly }
+
+ if (has_assembly){
+ ch_assembly = assembly.map { meta, assembly ->
+ [ meta.group, meta, assembly] }
+ .groupTuple(by: [0])
+ .map{ group, metas, assembly ->
+ def meta = [:]
+ meta.id = metas.group.unique().join()
+ meta.sample = metas.sample.join("_")
+ meta.flowcell = metas.flowcell.unique().join()
+ meta.group = metas.group.unique().join()
+ meta.assembly = metas.assembly.unique().join()
+ meta.type = metas.type.unique().join()
+ return [meta, assembly[0]] }
+ }
+
+ } else if (params.type.toUpperCase() == "SR") {
+ ch_reads_assembly = ch_reads
+ .map { meta, fastq ->
+ return [meta, fastq[0], fastq[1]] }
+
+ } else {
+ ch_reads_assembly = ch_reads
+ }
+
+ if (!has_assembly){
+ if (assembly_tool == 'metaspades') {
+ METASPADES(ch_reads_assembly)
+ ch_assembly = METASPADES.out.assembly
+ ch_assembler_v = METASPADES.out.v_metaspades
+ } else if (assembly_tool == 'megahit') {
+ MEGAHIT(ch_reads_assembly)
+ ch_assembly = MEGAHIT.out.assembly
+ ch_assembler_v = MEGAHIT.out.v_megahit
+ } else if (assembly_tool == 'hifiasm-meta') {
+ HIFIASM_META(ch_reads_assembly)
+ ch_assembly = HIFIASM_META.out.assembly
+ ch_assembler_v = HIFIASM_META.out.v_hifiasm_meta
+ } else if (assembly_tool == 'metaflye') {
+ METAFLYE(ch_reads_assembly)
ch_assembly = METAFLYE.out.assembly
ch_assembler_v = METAFLYE.out.v_metaflye
- }
- else {
+ } else {
exit 1, "Invalid assembly parameter: ${assembly_tool}"
- }
+ }
}
-
+
ch_assembly_renamed = RENAME_CONTIGS(ch_assembly)
ch_assembly_quast = ch_assembly_renamed
.map { meta, file -> file }
@@ -58,9 +131,22 @@ workflow STEP_02_ASSEMBLY {
ch_idxstats = Channel.empty()
ch_flagstat = Channel.empty()
- ch_reads = reads
+
+ if (params.coassembly){
+ ch_reads.map { meta, fastq ->
+ [ meta.group, meta, fastq ]}
+ .set { ch_reads_tmp }
+
+ ch_assembly_renamed.map { meta, assembly ->
+ [ meta.group, assembly ]}
+ .combine(ch_reads_tmp, by: 0)
+ .map { group, assembly, meta, fastq ->
+ [ meta, assembly,fastq ]}
+ .set { ch_contigs_and_reads }
+ } else {
+ ch_contigs_and_reads = ch_assembly_renamed.join(ch_reads, remainder: true)
+ }
- ch_contigs_and_reads = ch_assembly_renamed.join(ch_reads, remainder: true)
if ( params.type.toUpperCase() == "SR" ) {
READS_DEDUPLICATION ( ch_contigs_and_reads )
@@ -71,10 +157,10 @@ workflow STEP_02_ASSEMBLY {
ch_bwa_mem_v = READS_DEDUPLICATION.out.v_bwa_mem2
} else {
- MINIMAP2(ch_contigs_and_reads,"02_assembly/02_3_reads_vs_primary_assembly")
- ch_bam = MINIMAP2.out.bam
+ MINIMAP2(ch_contigs_and_reads,"02_assembly/02_3_reads_vs_primary_assembly")
+ ch_bam = MINIMAP2.out.bam
- ch_minimap2_v = MINIMAP2.out.v_minimap2
+ ch_minimap2_v = MINIMAP2.out.v_minimap2
}
diff --git a/subworkflows/03_filtering.nf b/subworkflows/03_filtering.nf
index 6ae3938..bf92a0b 100644
--- a/subworkflows/03_filtering.nf
+++ b/subworkflows/03_filtering.nf
@@ -23,10 +23,33 @@ workflow STEP_03_ASSEMBLY_FILTER {
ch_chunk_assembly_for_filter = assemblies
.splitFasta(by: 100000, file: true)
-
- ch_assembly_and_idxstats = ch_chunk_assembly_for_filter
+ if (params.coassembly){
+ idxstats.map { meta, idxstats ->
+ [ meta.group, meta, idxstats] }
+ .groupTuple(by: [0])
+ .map { group, metas, idxstats ->
+ def meta = [:]
+ meta.id = metas.group.unique().join()
+ meta.sample = metas.sample.join("_")
+ meta.flowcell = metas.flowcell.unique().join()
+ meta.group = metas.group.unique().join()
+ meta.assembly = metas.assembly.unique().join()
+ meta.type = metas.type.unique().join()
+ [ group, meta, idxstats] }
+ .set { ch_idxstats_tmp }
+ ch_chunk_assembly_for_filter.map { meta, assembly ->
+ [ meta.group, assembly ]}
+ .combine(ch_idxstats_tmp, by: 0)
+ .map { group, assembly, meta, idxstats ->
+ [ meta, assembly, idxstats ]}
+ .set { ch_assembly_and_idxstats }
+ } else {
+ ch_assembly_and_idxstats = ch_chunk_assembly_for_filter
.combine(idxstats, by:0)
+ }
+
+
CHUNK_ASSEMBLY_FILTER (
ch_assembly_and_idxstats,
@@ -52,69 +75,85 @@ workflow STEP_03_ASSEMBLY_FILTER {
ch_merged_selected = MERGE_ASSEMBLY_FILTER.out.merged_selected
discarded_contigs = MERGE_ASSEMBLY_FILTER.out.merged_discarded
+ ch_merged_selected_all = ch_merged_selected
+ .map { meta, file -> file }
+ .collect()
+ QUAST( ch_merged_selected_all, "${filtering_outdir}/03_1_filtered_assembly/" )
+ ch_quast_report = QUAST.out.report
- // Differentiate sample with and without discarded_contigs
- // samples with no discarded_contigs are not going to be processed to process
- discarded_contigs_category = discarded_contigs.branch{
- without: it[1].isEmpty()
- with: !it[1].isEmpty()
- }
- samples_without_discarded_contigs = discarded_contigs_category.without.map{ it -> it[0]}
- samples_with_discarded_contigs = discarded_contigs_category.with.map{ it -> it[0]}
+ // Differentiate sample with and without discarded_contigs
+ // samples with no discarded_contigs are not going to be processed to process
+ discarded_contigs_category = discarded_contigs.branch{
+ without: it[1].isEmpty()
+ with: !it[1].isEmpty()
+ }
-
- // make a symblink with the bam and bai from step 02 for samples that have not been affected by the filtering (no contig discarded)
- result_path_dir = file("${params.outdir}/${filtered_assembly_bam_outdir}/")
- result_path_dir.mkdirs()
- samples_without_discarded_contigs.map {
- sample -> { file("${result_path_dir}/${sample.id}/").mkdir()
- file("${params.outdir}/${unfiltered_assembly_bam_outdir}/${sample.id}/${sample.id}.sort.bam")
- .mklink("${result_path_dir}/${sample.id}/${sample.id}.sort.bam", overwrite:true)
- file("${params.outdir}/${unfiltered_assembly_bam_outdir}/${sample.id}/${sample.id}.sort.bam.bai")
- .mklink("${result_path_dir}/${sample.id}/${sample.id}.sort.bam.bai", overwrite:true)
- }
- }
-
- ch_bam_unchanged_by_filtering = samples_without_discarded_contigs.join(bam)
-
- ch_selected_contigs_and_reads = samples_with_discarded_contigs.join(ch_merged_selected).join(reads)
-
-
+ if (params.coassembly){
+ discarded_contigs_category.without.map { meta, discarded_empty -> [ meta.group ]}
+ .combine( bam.map { meta, bam, bai ->
+ [ meta.group, meta, bam, bai ]}, by: 0)
+ .map{ group, meta, bam, bai ->
+ [ meta, bam, bai ]}
+ .set{ ch_bam_unchanged_by_filtering }
+
+ ch_selected_contigs_and_reads= discarded_contigs_category.with.map {meta, discarded_contigs -> meta.group}
+ .join( ch_merged_selected.map { meta, contigs ->
+ [meta.group, meta, contigs]})
+ .combine( reads.map{ meta, reads ->
+ [ meta.group, meta, reads ]}, by: 0)
+ .map{ group, meta_contigs, contigs, meta_reads, reads ->
+ [ meta_reads, contigs, reads ]}
+
+ } else {
+ ch_bam_unchanged_by_filtering = discarded_contigs_category.without.map{ it -> it[0]}
+ .join(bam)
+
+ ch_selected_contigs_and_reads = discarded_contigs_category.with.map{ it -> it[0]}
+ .join(ch_merged_selected).join(reads)
+ }
+
+ // make a symblink with the bam and bai from step 02 for samples that have not been affected by the filtering (no contig discarded)
+ result_path_dir = file("${params.outdir}/${filtered_assembly_bam_outdir}/")
+ result_path_dir.mkdirs()
+
+ ch_bam_unchanged_by_filtering.map { meta, bam, bai ->
+ { file("${result_path_dir}/${meta.id}/").mkdir()
+ file("${params.outdir}/${unfiltered_assembly_bam_outdir}/${meta.id}/${meta.id}.bam")
+ .mklink("${result_path_dir}/${meta.id}/${meta.id}.bam", overwrite:true)
+ file("${params.outdir}/${unfiltered_assembly_bam_outdir}/${meta.id}/${meta.id}.bam.bai")
+ .mklink("${result_path_dir}/${meta.id}/${meta.id}.bam.bai", overwrite:true)
+ }
+ }
- if ( params.type.toUpperCase() == "SR" ) {
- BWA_MEM(ch_selected_contigs_and_reads, filtered_assembly_bam_outdir)
- ch_bam_post_filtering = BWA_MEM.out.bam
- }
- else {
- MINIMAP2(ch_selected_contigs_and_reads, filtered_assembly_bam_outdir)
- ch_bam_post_filtering = MINIMAP2.out.bam
- }
+ if ( params.type.toUpperCase() == "SR" ) {
+ BWA_MEM(ch_selected_contigs_and_reads, filtered_assembly_bam_outdir)
+ ch_bam_post_filtering = BWA_MEM.out.bam
+ }
+ else {
+ MINIMAP2(ch_selected_contigs_and_reads, filtered_assembly_bam_outdir)
+ ch_bam_post_filtering = MINIMAP2.out.bam
+ }
- ch_all_bam = samples_without_discarded_contigs.join(bam).mix(ch_bam_post_filtering)
+ ch_all_bam = ch_bam_unchanged_by_filtering.mix(ch_bam_post_filtering)
- GET_ALIGNMENT_METRICS(ch_all_bam, filtered_assembly_bam_outdir)
+ GET_ALIGNMENT_METRICS(ch_all_bam, filtered_assembly_bam_outdir)
- ch_flagstat = GET_ALIGNMENT_METRICS.out.sam_flagstat
- ch_coverage = GET_ALIGNMENT_METRICS.out.sam_coverage
+ ch_flagstat = GET_ALIGNMENT_METRICS.out.sam_flagstat
+ ch_coverage = GET_ALIGNMENT_METRICS.out.sam_coverage
- ch_merged_quast = ch_merged_selected
- .map { meta, file -> file }
- .collect()
- QUAST( ch_merged_quast, "${filtering_outdir}/03_1_filtered_assembly/" )
- ch_quast_report = QUAST.out.report
+
- ch_final_bam = ch_bam_post_filtering.mix(ch_bam_unchanged_by_filtering)
- emit:
- selected_contigs = ch_merged_selected
- quast_report = ch_quast_report
- bam = ch_final_bam
- sam_coverage = ch_coverage
- sam_flagstat = ch_flagstat
+ emit:
+ selected_contigs = ch_merged_selected
+ quast_report = ch_quast_report
+ bam = ch_all_bam
+ sam_coverage = ch_coverage
+ sam_flagstat = ch_flagstat
}
diff --git a/subworkflows/04_structural_annot.nf b/subworkflows/04_structural_annot.nf
index 49841ef..505e633 100644
--- a/subworkflows/04_structural_annot.nf
+++ b/subworkflows/04_structural_annot.nf
@@ -17,8 +17,6 @@ workflow STEP_04_STRUCTURAL_ANNOT {
MERGE_ANNOTATIONS(annotations_ch)
- ch_software_versions = PRODIGAL.out.v_prodigal.first()
-
ch_software_versions = PRODIGAL.out.v_prodigal.first().mix( BARRNAP.out.v_barrnap.first(),
TRNASCAN_SE.out.v_tRNAscan.first())
diff --git a/subworkflows/05_protein_alignment.nf b/subworkflows/05_protein_alignment.nf
index 56258c4..b178755 100644
--- a/subworkflows/05_protein_alignment.nf
+++ b/subworkflows/05_protein_alignment.nf
@@ -13,7 +13,7 @@ workflow STEP_05_PROTEIN_ALIGNMENT {
DIAMOND (
prokka_faa,
- diamond,
+ diamond
)
ch_diamond_result = DIAMOND.out.m8
diff --git a/subworkflows/06_functionnal_annot.nf b/subworkflows/06_functionnal_annot.nf
index 5b86055..ad48129 100644
--- a/subworkflows/06_functionnal_annot.nf
+++ b/subworkflows/06_functionnal_annot.nf
@@ -28,7 +28,22 @@ workflow STEP_06_FUNC_ANNOT {
ch_global_clstr_table = CD_HIT.out.global_clstr_table
ch_cdhit_v = CD_HIT.out.v_cdhit
- QUANTIFICATION ( gff, bam, ch_individual_clstr_table, ch_global_clstr_table)
+ if (params.coassembly){
+ bam.map { meta, bam, bai ->
+ [ meta.group, meta, bam, bai ]}
+ .set { ch_bam_tmp }
+
+ gff.map { meta, gff ->
+ [ meta.group, gff]}
+ .combine( ch_bam_tmp, by: 0)
+ .map { group, gff, meta, bam, bai ->
+ [ meta, gff, bam, bai ]}
+ .set { ch_gff_and_bam }
+ } else {
+ ch_gff_and_bam = gff.join(bam, remainder: false)
+ }
+
+ QUANTIFICATION ( ch_gff_and_bam, ch_individual_clstr_table, ch_global_clstr_table)
ch_quant_table = QUANTIFICATION.out.quantification_table
ch_quant_report = QUANTIFICATION.out.quant_report
ch_featurecounts_v = QUANTIFICATION.out.v_featurecounts
diff --git a/subworkflows/07_taxonomic_affi.nf b/subworkflows/07_taxonomic_affi.nf
index 99ead6c..4f1eb9b 100644
--- a/subworkflows/07_taxonomic_affi.nf
+++ b/subworkflows/07_taxonomic_affi.nf
@@ -8,7 +8,20 @@ workflow STEP_07_TAXO_AFFI {
diamond_result // channel: [ val(meta), path(diamond_file) ]
sam_coverage // channel: [ val(meta), path(samtools coverage) ]
main:
- ch_assign_taxo_input = diamond_result.join(sam_coverage, remainder: true)
+ if (params.coassembly){
+ sam_coverage.map { meta, cov ->
+ [ meta.group, meta, cov ]}
+ .set { ch_sam_cov_tmp }
+
+ diamond_result.map { meta, m8 ->
+ [ meta.group, m8]}
+ .combine( ch_sam_cov_tmp, by: 0)
+ .map { group, m8, meta, cov ->
+ [ meta, m8, cov ]}
+ .set { ch_assign_taxo_input }
+ } else {
+ ch_assign_taxo_input = diamond_result.join(sam_coverage, remainder: true)
+ }
ASSIGN_TAXONOMY ( taxonomy, ch_assign_taxo_input )
diff --git a/subworkflows/08_binning.nf b/subworkflows/08_binning.nf
index 59b9c7b..32b9e32 100644
--- a/subworkflows/08_binning.nf
+++ b/subworkflows/08_binning.nf
@@ -52,21 +52,21 @@ workflow STEP_08_BINNING {
if (params.binning_cross_alignment == 'all') {
// combine assemblies with reads of all samples
ch_reads_assembly = reads.combine(assembly)
- .map{ meta_reads, reads, meta_assembly, assembly ->
- if (meta_reads != meta_assembly){
- [[id:meta_reads.id+"_"+meta_assembly.id, sample:meta_assembly.sample, flowcell:meta_assembly.flowcell, group:meta_assembly.group, assembly:meta_assembly.assembly, type:meta_assembly.type], assembly, reads]
- }
- }
+ .map{ meta_reads, reads, meta_assembly, assembly ->
+ if (((meta_reads.id != meta_assembly.id) && !(params.coassembly)) || (params.coassembly && (meta_reads.group != meta_assembly.group))){
+ [[id:meta_reads.id+"_"+meta_assembly.id, sample:meta_assembly.sample, flowcell:meta_assembly.flowcell, group:meta_assembly.group, assembly:meta_assembly.assembly, type:meta_assembly.type], assembly, reads]
+ }
+ }
} else if (params.binning_cross_alignment == 'group'){
// combine assemblies with reads of samples from same group
ch_assembly_group = assembly.map{ meta, assembly -> [ meta.group, meta, assembly ] }
ch_reads_assembly = reads.map{ meta, reads -> [ meta.group, meta, reads ] }
- .combine(ch_assembly_group, by: 0)
- .map{ group, meta_reads, reads, meta_assembly, assembly ->
- if (meta_reads != meta_assembly){
- [[id:meta_reads.id+"_"+meta_assembly.id, sample:meta_assembly.sample, flowcell:meta_assembly.flowcell, group:meta_assembly.group, assembly:meta_assembly.assembly, type:meta_assembly.type], assembly, reads]
- }
- }
+ .combine(ch_assembly_group, by: 0)
+ .map{ group, meta_reads, reads, meta_assembly, assembly ->
+ if (meta_reads != meta_assembly){
+ [[id:meta_reads.id+"_"+meta_assembly.id, sample:meta_assembly.sample, flowcell:meta_assembly.flowcell, group:meta_assembly.group, assembly:meta_assembly.assembly, type:meta_assembly.type], assembly, reads]
+ }
+ }
}
// cross alignment
if (params.type == 'SR') {
@@ -78,21 +78,51 @@ workflow STEP_08_BINNING {
}
// formatting channel
ch_bam = ch_bam_cross_alignment.mix(bam)
- .map { meta, bam, bai -> [ meta.sample, meta, bam, bai ] }
- .groupTuple(by: [0])
- .map { sample,metas, bam, bai ->
- [ metas.min { it.id.size() }, bam, bai ]
- }
+ .map { meta, bam, bai ->
+ if (params.coassembly){[ meta.group, meta, bam, bai ]}
+ else {[ meta.sample, meta, bam, bai ]}}
+ .groupTuple(by: [0])
+ .map { sample,metas, bam, bai ->
+ [ metas.min { it.id.size() }, bam, bai ]
+ }
} else {
ch_bam = bam
}
+ if (params.coassembly){
+ if (params.binning_cross_alignment == 'all') {
+ ch_bam.map { meta, bam, bai -> [ meta.group, bam, bai ] }
+ .set { ch_bam_tmp }
+ } else {
+ ch_bam.map { meta, bam, bai -> [ meta.group, meta, bam, bai ] }
+ .groupTuple(by: [0])
+ .map { group, metas, bam, bai ->
+ [ group, bam, bai ]}
+ .set { ch_bam_tmp }
+ }
+ assembly.map { meta, assembly ->
+ [ meta.group, meta, assembly ]}
+ .combine( ch_bam_tmp, by: 0)
+ .map { group, meta, assembly, bam, bai ->
+ [ meta, assembly, bam, bai]}
+ .tap { ch_assembly_bam }
+ .map { meta, assembly, bam, bai ->
+ [ meta, assembly ]}
+ .tap { ch_assembly }
+
+ ch_assembly_bam.map{ meta, assembly, bam, bai ->
+ [ meta, bam, bai ]}
+ .set{ ch_bam}
+ } else {
+ ch_assembly_bam = assembly.join(ch_bam)
+ ch_assembly = assembly
+ }
///////////
/// BINNING
///////////
ch_depth = GENERATE_DEPTH_FILES(ch_bam)
- ch_assembly_depth = assembly.join(ch_depth)
+ ch_assembly_depth = ch_assembly.join(ch_depth)
METABAT2(ch_assembly_depth)
ch_metabat_bins = METABAT2.out.bins.filter{ t -> t[1].list().size()}
@@ -102,8 +132,6 @@ workflow STEP_08_BINNING {
ch_maxbin_bins = MAXBIN2.out.bins.filter{ t -> t[1].list().size()}
ch_maxbin_v = MAXBIN2.out.v_maxbin
- ch_assembly_bam = assembly.join(ch_bam)
-
CONCOCT(ch_assembly_bam)
ch_concoct_bins = CONCOCT.out.bins.filter{ t -> t[1].list().size()}
ch_concoct_v = CONCOCT.out.v_concoct
@@ -120,7 +148,8 @@ workflow STEP_08_BINNING {
(it[2] != null && it[3] != null)
single: true
}
-
+
+ //bins set with bins form at least 2 binners
ch_bins_multiple = ch_bins_set.multiple.map{ meta, bins1, bins2, bins3 ->
if ( bins1 == null ) {
return [meta, bins2, bins3, bins1]
@@ -131,6 +160,7 @@ workflow STEP_08_BINNING {
}
}
+ //bins set with bins form only 1 binner
ch_bins_single = ch_bins_set.single.map{ meta, bins1, bins2, bins3 ->
if ( bins1 != null ) {
return [meta, bins1]
@@ -160,7 +190,7 @@ workflow STEP_08_BINNING {
.map { meta, file -> file}
.collect()
- ch_bins_assembly = METAWRAP_REFINMENT.out.bins.join(assembly)
+ ch_bins_assembly = METAWRAP_REFINMENT.out.bins.join(ch_assembly)
UNBINNED_CONTIGS(ch_bins_assembly)
--
GitLab
From 55cd96723438aba1ef1498ef84e1fab2ac4dd5a4 Mon Sep 17 00:00:00 2001
From: "maina.vienne" <maina.vienne@inrae.fr>
Date: Wed, 11 Jan 2023 11:40:16 +0100
Subject: [PATCH 174/194] Fix add quast info for bin stats
---
main.nf | 2 ++
modules/sum_up_bins_informations.nf | 10 +++++++++-
subworkflows/08_binning.nf | 2 +-
3 files changed, 12 insertions(+), 2 deletions(-)
diff --git a/main.nf b/main.nf
index ac14614..7a022ef 100644
--- a/main.nf
+++ b/main.nf
@@ -446,6 +446,8 @@ workflow {
}
if ( !params.stop_at_clean && !params.stop_at_assembly && !params.stop_at_filtering && !params.stop_at_structural_annot && !params.skip_binning ) {
+ ch_quast=ch_quast_after_filter_report.ifEmpty(ch_quast_before_filter_report)
+
S08_BINNING( ch_reads, ch_assembly, ch_bam, ch_gtbdtk_db, ch_quast )
ch_bins_abundances_report = S08_BINNING.out.bins_abundances_report
ch_bins_stats_report = S08_BINNING.out.bins_stats_report
diff --git a/modules/sum_up_bins_informations.nf b/modules/sum_up_bins_informations.nf
index 842b43c..5cfc103 100644
--- a/modules/sum_up_bins_informations.nf
+++ b/modules/sum_up_bins_informations.nf
@@ -44,8 +44,16 @@ process ADD_QUAST_INFO_TO_BINS {
script:
"""
+ awk -F"\t" '{
+ if (NR==1) {
+ val=-1;
+ for(i=1;i<=NF;i++) {
+ if (\$i ~ /${meta.id}.*/) {
+ val=i;}}}
+ if(val != -1) print \$1 "\t" \$val} ' report.tsv > report_${meta.id}.tsv
+
add_info_to_metawrap_stat.py -s $bins_stat \
- -q $quast_report \
+ -q report_${meta.id}.tsv \
-o "${meta.id}_bins_stat_and_quality.tsv"
"""
}
diff --git a/subworkflows/08_binning.nf b/subworkflows/08_binning.nf
index 32b9e32..69a7962 100644
--- a/subworkflows/08_binning.nf
+++ b/subworkflows/08_binning.nf
@@ -194,7 +194,7 @@ workflow STEP_08_BINNING {
UNBINNED_CONTIGS(ch_bins_assembly)
- ch_metawrap_stats_and_quast = METAWRAP_REFINMENT.out.checkm_stats.join(quast)
+ ch_metawrap_stats_and_quast = METAWRAP_REFINMENT.out.checkm_stats.combine(quast)
ch_metawrap_v = METAWRAP_REFINMENT.out.v_metawrap
ADD_QUAST_INFO_TO_BINS(ch_metawrap_stats_and_quast)
--
GitLab
From 5bd0f3a9ba435c3b4d681413a57758c77df46f9b Mon Sep 17 00:00:00 2001
From: Vincent Darbot <vincent.darbot@inrae.fr>
Date: Fri, 13 Jan 2023 12:02:20 +0100
Subject: [PATCH 175/194] refactoring bin/merge_kaiju_results.py
---
bin/merge_kaiju_results.py | 116 +++++++++++++++++++++---------------
bin/rename_contigs.py | 12 ++++
main.nf | 3 +
modules/assembly.nf | 26 ++++++++
subworkflows/02_assembly.nf | 13 +++-
5 files changed, 122 insertions(+), 48 deletions(-)
diff --git a/bin/merge_kaiju_results.py b/bin/merge_kaiju_results.py
index 1c8994d..016d037 100755
--- a/bin/merge_kaiju_results.py
+++ b/bin/merge_kaiju_results.py
@@ -28,57 +28,79 @@ from datetime import datetime
# Manage parameters.
-parser = argparse.ArgumentParser(description = 'Script which join \
-kaiju results by level of taxonomy of interest for all samples.')
+def parse_args():
+ parser = argparse.ArgumentParser(description = 'Script which join \
+ kaiju results by level of taxonomy of interest for all samples.')
-parser.add_argument('-f', '--list_of_kaiju_files', required = True, \
-help = 'List of kaiju summary files by the level \
-of taxonomy of interest .')
+ parser.add_argument('-f', '--list_of_kaiju_files', required = True, \
+ help = 'List of kaiju summary files by the level \
+ of taxonomy of interest .')
-parser.add_argument('-o', '--output_file', required = True, \
-help = 'Name of output file containing counts \
-and percentage of reads in each sample for each element \
-of the level of taxonomy of interest.')
+ parser.add_argument('-o', '--output_file', required = True, \
+ help = 'Name of output file containing counts \
+ and percentage of reads in each sample for each element \
+ of the level of taxonomy of interest.')
-parser.add_argument('-v', '--version', action = 'version', \
-version = __version__)
+ parser.add_argument('-v', '--version', action = 'version', \
+ version = __version__)
-args = parser.parse_args()
+ args = parser.parse_args()
-# Recovery of the list of annotations files.
-with open(args.list_of_kaiju_files) as fkaiju_list:
- kaiju_files = fkaiju_list.read().split()
+ return args
-# Merge kaiju results for all samples.
-for (kaiju_idx,kaiju_path) in enumerate(sorted(kaiju_files)):
- print(kaiju_idx)
- if(kaiju_idx==0):
- merge = pd.read_csv(kaiju_path, delimiter='\t', dtype=str)
- else:
- if(kaiju_idx==1):
- sample_name = merge.iloc[0,0].split('_kaiju_MEM_verbose.out')
- merge.drop('file', inplace=True, axis=1)
+
+def retrieve_annotations_files(list_kaiju_files):
+ '''
+ Recovery of the list of annotations files.
+ '''
+ with open(list_kaiju_files) as fkaiju_list:
+ kaiju_files = fkaiju_list.read().split()
+ return kaiju_files
+
+
+def merge_kaiju_files(kaiju_files):
+ '''
+ Merge kaiju results for all samples.
+ '''
+ for (kaiju_idx,kaiju_path) in enumerate(sorted(kaiju_files)):
+ if(kaiju_idx==0):
+ merge = pd.read_csv(kaiju_path, delimiter='\t', dtype=str)
else:
- sample_name = kaiju_results.iloc[0,0].split('_kaiju_MEM_verbose.out')
- merge.drop('file', inplace=True, axis=1)
- kaiju_results = pd.read_csv(kaiju_path, delimiter='\t', dtype=str)
- merge = pd.merge(merge,kaiju_results,left_on="taxon_name",\
- right_on='taxon_name', how='outer', suffixes=('_'+sample_name[0],''))
- merge['taxon_id'] = merge['taxon_id'].fillna(merge['taxon_id_' + sample_name[0]])
- merge.drop(['taxon_id_' + sample_name[0]], inplace=True, axis=1)
-
-# Rename columns corresponding to the last kaiju file (only if number of files > 1)
-if(kaiju_idx>0):
- sample_name = kaiju_results.iloc[0,0].split('_kaiju_MEM_verbose.out')
-else:
- sample_name = merge.iloc[0,0].split('_kaiju_MEM_verbose.out')
-merge.rename(columns = {'percent': 'percent_' + sample_name[0], \
-'reads': 'reads_' + sample_name[0]},inplace=True)
-merge.drop('file', inplace=True, axis=1)
-merge = merge[['taxon_name', 'taxon_id'] + [col for col in merge if (col != 'taxon_name' and col != 'taxon_id')]]
-
-# Fill the NaN by 0.
-merge.fillna(0, inplace=True)
-
-# Write merge data frame in output file.
-merge.to_csv(args.output_file, sep="\t", index=False)
+ if(kaiju_idx==1):
+ sample_name = merge.iloc[0,0].split('_kaiju_MEM_verbose.out')
+ merge.drop('file', inplace=True, axis=1)
+ else:
+ sample_name = kaiju_results.iloc[0,0].split('_kaiju_MEM_verbose.out')
+ merge.drop('file', inplace=True, axis=1)
+ kaiju_results = pd.read_csv(kaiju_path, delimiter='\t', dtype=str)
+ merge = pd.merge(merge,kaiju_results,left_on="taxon_name",\
+ right_on='taxon_name', how='outer', suffixes=('_'+sample_name[0],''))
+ merge['taxon_id'] = merge['taxon_id'].fillna(merge['taxon_id_' + sample_name[0]])
+ merge.drop(['taxon_id_' + sample_name[0]], inplace=True, axis=1)
+
+ # Rename columns corresponding to the last kaiju file (only if number of files > 1)
+ if(kaiju_idx>0):
+ sample_name = kaiju_results.iloc[0,0].split('_kaiju_MEM_verbose.out')
+ else:
+ sample_name = merge.iloc[0,0].split('_kaiju_MEM_verbose.out')
+
+ merge.rename(columns = {'percent': 'percent_' + sample_name[0], \
+ 'reads': 'reads_' + sample_name[0]},inplace=True)
+
+ merge.drop('file', inplace=True, axis=1)
+ merge = merge[['taxon_name', 'taxon_id'] + [col for col in merge if (col != 'taxon_name' and col != 'taxon_id')]]
+ # Fill the NaN by 0.
+ merge.fillna(0, inplace=True)
+
+ return merge
+
+
+def main():
+ args = parse_args()
+ kaiju_files = retrieve_annotations_files(args.list_of_kaiju_files)
+ merge = merge_kaiju_files(kaiju_files)
+ merge.to_csv(args.output_file, sep="\t", index=False)
+
+
+if __name__ == "__main__":
+ main()
\ No newline at end of file
diff --git a/bin/rename_contigs.py b/bin/rename_contigs.py
index 264d9cf..beb627b 100755
--- a/bin/rename_contigs.py
+++ b/bin/rename_contigs.py
@@ -67,5 +67,17 @@ def main():
for i, (name, seq) in enumerate(pyfastx.Fasta(fna_file, build_index=False)):
fh_fna.write(f'>{sample}_c{i+1} {name}\n{seq}\n')
+ sample = args.sample
+ fna_file = args.fna_file
+ out_fna = args.out_fna
+ old2new_contig_name = "old_to_new_contig_name.tsv"
+
+
+ logging.info(f'Writting renamed fasta file in {out_fna}')
+ with open(out_fna, "w") as fh_fna, open(old2new_contig_name, 'w') as mout:
+ for i, (name, seq) in enumerate(pyfastx.Fasta(fna_file, build_index=False)):
+ fh_fna.write(f'>{sample}_c{i+1} {name}\n{seq}\n')
+ mout.write(f'{name}\t{sample}_c{i+1}\n')
+
if __name__ == '__main__':
main()
\ No newline at end of file
diff --git a/main.nf b/main.nf
index 7a022ef..b42462b 100644
--- a/main.nf
+++ b/main.nf
@@ -376,6 +376,9 @@ workflow {
ch_quast_before_filter_report = S02_ASSEMBLY.out.assembly_report
ch_software_versions_S02 = S02_ASSEMBLY.out.software_versions
+ if (params.type.toUpperCase() == 'HIFI') {
+ ch_circular = S02_ASSEMBLY.out.circular
+ }
}
if ( !params.stop_at_clean && !params.stop_at_assembly && !params.skip_filtering ) {
diff --git a/modules/assembly.nf b/modules/assembly.nf
index 5c24b43..0b4d56f 100644
--- a/modules/assembly.nf
+++ b/modules/assembly.nf
@@ -106,6 +106,7 @@ process METAFLYE {
output:
tuple val(meta), path("metaflye_${meta.id}/${meta.id}.contigs.fa"), emit: assembly
tuple val(meta.id), path("metaflye_${meta.id}/${meta.id}.log"), path("metaflye_${meta.id}/${meta.id}.params.json"), emit: report
+ tuple val(meta.id), path("metaflye_${meta.id}/assembly_info.txt"), emit: infos
path "v_metaflye.txt", emit: v_metaflye
script:
@@ -121,6 +122,31 @@ process METAFLYE {
"""
}
+process CIRCULAR_CONTIGS {
+ tag "${meta.id}"
+ publishDir "${params.outdir}/02_assembly/02_1_primary_assembly", mode: 'copy'
+ label 'PYTHON'
+
+ input:
+ val(assembler)
+ tuple val(meta), path("${meta.id}.fna")
+ path(infos)
+
+ output:
+ tuple val(meta), path("${meta.id}.circular.contigs.fna"), emit: circular
+
+ script:
+ """
+ if [ $assembler == 'metaflye' ]
+ then
+ retrieve_circular_contigs.py -a metaflye -f ${meta.id}.fna -i $infos -o ${meta.id}.circular.contigs.fna
+ elif [ $assembler == 'hifiasm-meta' ]
+ then
+ retrieve_circular_contigs.py -a hifiasm-meta -f ${meta.id}.fna -o ${meta.id}.circular.contigs.fna
+ fi
+ """
+}
+
process RENAME_CONTIGS {
tag "${meta.id}"
publishDir "${params.outdir}/02_assembly/02_1_primary_assembly", mode: 'copy'
diff --git a/subworkflows/02_assembly.nf b/subworkflows/02_assembly.nf
index 46c4da5..ba94a90 100644
--- a/subworkflows/02_assembly.nf
+++ b/subworkflows/02_assembly.nf
@@ -1,4 +1,4 @@
-include { METASPADES; MEGAHIT; HIFIASM_META; METAFLYE; RENAME_CONTIGS } from '../modules/assembly'
+include { METASPADES; MEGAHIT; HIFIASM_META; METAFLYE; CIRCULAR_CONTIGS; RENAME_CONTIGS } from '../modules/assembly'
include { QUAST as ASSEMBLY_QUAST} from '../modules/metaquast'
include { READS_DEDUPLICATION } from '../modules/reads_deduplication'
include { MINIMAP2 } from '../modules/read_alignment'
@@ -99,6 +99,7 @@ workflow STEP_02_ASSEMBLY {
}
if (!has_assembly){
+ ch_circular = Channel.empty()
if (assembly_tool == 'metaspades') {
METASPADES(ch_reads_assembly)
ch_assembly = METASPADES.out.assembly
@@ -108,12 +109,14 @@ workflow STEP_02_ASSEMBLY {
ch_assembly = MEGAHIT.out.assembly
ch_assembler_v = MEGAHIT.out.v_megahit
} else if (assembly_tool == 'hifiasm-meta') {
+ ch_infos = Channel.empty()
HIFIASM_META(ch_reads_assembly)
ch_assembly = HIFIASM_META.out.assembly
ch_assembler_v = HIFIASM_META.out.v_hifiasm_meta
} else if (assembly_tool == 'metaflye') {
METAFLYE(ch_reads_assembly)
ch_assembly = METAFLYE.out.assembly
+ ch_infos = METAFLYE.out.infos
ch_assembler_v = METAFLYE.out.v_metaflye
} else {
exit 1, "Invalid assembly parameter: ${assembly_tool}"
@@ -125,6 +128,13 @@ workflow STEP_02_ASSEMBLY {
.map { meta, file -> file }
.collect()
+ if ( params.type.toUpperCase() == "HIFI" ) {
+
+ CIRCULAR_CONTIGS(assembly_tool, ch_reads_assembly, ch_infos)
+ ch_circular = CIRCULAR_CONTIGS.out.circular
+
+ }
+
ASSEMBLY_QUAST( ch_assembly_quast,"02_assembly/02_1_primary_assembly/")
ch_assembly_report = ASSEMBLY_QUAST.out.report
ch_quast_v = ASSEMBLY_QUAST.out.v_quast
@@ -179,6 +189,7 @@ workflow STEP_02_ASSEMBLY {
emit:
assembly = ch_assembly_renamed
+ circular = ch_circular
reads = ch_reads
bam = ch_bam
--
GitLab
From ee77858b9dac37227279a022ccca25613cc4b209 Mon Sep 17 00:00:00 2001
From: Vincent Darbot <vincent.darbot@inrae.fr>
Date: Fri, 13 Jan 2023 15:15:40 +0100
Subject: [PATCH 176/194] refactoring bin/merge_kaiju_results.py
---
bin/merge_kaiju_results.py | 1 -
1 file changed, 1 deletion(-)
diff --git a/bin/merge_kaiju_results.py b/bin/merge_kaiju_results.py
index 016d037..6d21cb4 100755
--- a/bin/merge_kaiju_results.py
+++ b/bin/merge_kaiju_results.py
@@ -26,7 +26,6 @@ import sys
import pandas as pd
from datetime import datetime
-
# Manage parameters.
def parse_args():
parser = argparse.ArgumentParser(description = 'Script which join \
--
GitLab
From 814ddf913907a68b4e7a81de0d74521aebfa1b5e Mon Sep 17 00:00:00 2001
From: Vincent Darbot <vincent.darbot@inrae.fr>
Date: Sat, 14 Jan 2023 11:59:02 +0100
Subject: [PATCH 177/194] IN PROGRESS: circular contigs
---
bin/retrieve_circular_contigs.py | 61 ++++++++++++++++++++++++++++++++
subworkflows/02_assembly.nf | 3 +-
2 files changed, 62 insertions(+), 2 deletions(-)
create mode 100755 bin/retrieve_circular_contigs.py
diff --git a/bin/retrieve_circular_contigs.py b/bin/retrieve_circular_contigs.py
new file mode 100755
index 0000000..565190f
--- /dev/null
+++ b/bin/retrieve_circular_contigs.py
@@ -0,0 +1,61 @@
+#!/usr/bin/env python3
+
+## Imports
+from Bio import SeqIO
+import pandas as pd
+import argparse
+
+## Functions
+
+def parse_metaflye(input_fasta, info_file, circular_fasta):
+ """
+ metaflye fasta contains no information.
+ This is contained in the assembly_info.txt file.
+ """
+ df = pd.read_csv(info_file, sep='\t')
+ # this file may have changed symbols for yes, possibilities include a Y or +
+ circular_contigs = list(df[df['circ.'].isin(['Y', '+'])]["#seq_name"])
+
+ with open(circular_fasta, 'wt') as fh_out:
+ for rec in SeqIO.parse(input_fasta, "fasta"):
+ if rec.id in circular_contigs:
+ fh_out.write(rec.format("fasta"))
+
+def parse_hifiasm(input_fasta, circular_fasta):
+ """
+ hifiasm-meta circular contig names will have an "c" suffix.
+ """
+ with open(circular_fasta, 'wt') as fh_out:
+ for rec in SeqIO.parse(input_fasta, "fasta"):
+ if rec.id.endswith('c'):
+ fh_out.write(rec.format("fasta"))
+
+def main():
+ # Manage parameters
+ parser = argparse.ArgumentParser( description='Retrieve circular contigs from long-reads assemblies.')
+ # Inputs
+ group_input = parser.add_argument_group( 'Inputs' )
+ group_input.add_argument('-a', '--assembler', required=True, choices=['metaflye', 'hifiasm-meta'], help='Assembler where the assemblies come from.')
+ group_input.add_argument('-f', '--input-fasta', required=True, help='The path of assembly fasta file.')
+
+ group_input_otu_table = parser.add_argument_group( ' Metaflye ' )
+ group_input_otu_table.add_argument('-i','--info-file', default=None, help="The path of metaflye asembly info file.")
+ # output
+ group_output = parser.add_argument_group( 'Outputs' )
+ group_output.add_argument('-o','--output-fasta', default='circular_contigs.fna', help="The path of circular contigs fasta file. [Default: %(default)s]" )
+
+ args = parser.parse_args()
+
+ # Check for inputs
+ if args.assembler == "metaflye":
+ if args.info_file is None:
+ parser.error("\n\n#ERROR : --info-file is required with metaflye assembler.")
+ else:
+ parse_metaflye(args.input_fasta, args.info_file, args.output_fasta)
+
+ elif args.assembler == "hifiasm-meta":
+ parse_hifiasm(args.input_fasta, args.output_fasta)
+
+
+if __name__ == '__main__':
+ main()
\ No newline at end of file
diff --git a/subworkflows/02_assembly.nf b/subworkflows/02_assembly.nf
index ba94a90..b53a0d3 100644
--- a/subworkflows/02_assembly.nf
+++ b/subworkflows/02_assembly.nf
@@ -129,8 +129,7 @@ workflow STEP_02_ASSEMBLY {
.collect()
if ( params.type.toUpperCase() == "HIFI" ) {
-
- CIRCULAR_CONTIGS(assembly_tool, ch_reads_assembly, ch_infos)
+ CIRCULAR_CONTIGS(assembly_tool, ch_assembly_renamed, ch_infos)
ch_circular = CIRCULAR_CONTIGS.out.circular
}
--
GitLab
From fd55b25067532381419f86a9c3d58546c2d66c61 Mon Sep 17 00:00:00 2001
From: Maina Vienne <maina.vienne@inrae.fr>
Date: Mon, 16 Jan 2023 16:30:43 +0100
Subject: [PATCH 178/194] Replace metawrap bin_refinement with binette
---
README.md | 2 +-
...tawrap_stat.py => add_info_to_bin_stat.py} | 6 +-
bin/bin_refinement.sh | 549 ------------------
bin/metawrap/add_metrics_to_checkm.py | 134 -----
bin/metawrap/binning_refiner.py | 269 ---------
.../consolidate_multiple_bins_sets.py | 260 ---------
bin/metawrap/dereplicate_contigs.py | 74 ---
bin/metawrap/dereplicate_contigs_in_bins.py | 150 -----
bin/metawrap/get_contig_length.py | 73 ---
bin/metawrap/plot_binning_results.py | 246 --------
bin/metawrap/print_comment.py | 36 --
.../setup_indermediate_checkm2_files.py | 171 ------
conf/base.config | 2 +-
conf/test.config | 2 +-
docs/output.md | 2 +-
env/Singularity_recipe_binning | 18 +-
env/binning.yml | 21 +-
modules/binning.nf | 35 +-
modules/checkm2.nf | 2 +-
modules/get_db_versions.nf | 2 +-
modules/sum_up_bins_informations.nf | 2 +-
subworkflows/08_binning.nf | 82 +--
22 files changed, 83 insertions(+), 2055 deletions(-)
rename bin/{add_info_to_metawrap_stat.py => add_info_to_bin_stat.py} (96%)
delete mode 100755 bin/bin_refinement.sh
delete mode 100755 bin/metawrap/add_metrics_to_checkm.py
delete mode 100755 bin/metawrap/binning_refiner.py
delete mode 100755 bin/metawrap/consolidate_multiple_bins_sets.py
delete mode 100755 bin/metawrap/dereplicate_contigs.py
delete mode 100755 bin/metawrap/dereplicate_contigs_in_bins.py
delete mode 100755 bin/metawrap/get_contig_length.py
delete mode 100755 bin/metawrap/plot_binning_results.py
delete mode 100755 bin/metawrap/print_comment.py
delete mode 100644 bin/metawrap/setup_indermediate_checkm2_files.py
diff --git a/README.md b/README.md
index 1a3736b..b15d396 100644
--- a/README.md
+++ b/README.md
@@ -41,7 +41,7 @@ Many of these steps are optional and their necessity depends on the desired anal
* aligns reads samples against assemblies (according to the strategy used) ([BWA-MEM2](https://github.com/bwa-mem2/bwa-mem2) or [Minimap2](https://github.com/lh3/minimap2))
* performs metagenome binning ([METABAT2](https://bitbucket.org/berkeleylab/metabat/src/master/) + [MAXBIN2](https://sourceforge.net/projects/maxbin/) + [CONCOCT](https://github.com/BinPro/CONCOCT))
- * refines bin sets ([bin_refinement.sh](bin/bin_refinement.sh) adapt from [METAWRAP](https://github.com/bxlab/metaWRAP) bin_refinement)
+ * refines bin sets ([BINETTE](https://forgemia.inra.fr/jean.mainguy/binette/))
* dereplicates bins between samples ([DREP](https://github.com/MrOlm/drep))
* taxonomically affiliates the bins ([GTDBTK](https://github.com/Ecogenomics/GTDBTk))
* calculates bins abundances between samples ([BWA-MEM2](https://github.com/bwa-mem2/bwa-mem2) or [Minimap2](https://github.com/lh3/minimap2) + [SAMTOOLS](http://www.htslib.org/))
diff --git a/bin/add_info_to_metawrap_stat.py b/bin/add_info_to_bin_stat.py
similarity index 96%
rename from bin/add_info_to_metawrap_stat.py
rename to bin/add_info_to_bin_stat.py
index 3e6ff74..5389c9d 100755
--- a/bin/add_info_to_metawrap_stat.py
+++ b/bin/add_info_to_bin_stat.py
@@ -32,7 +32,7 @@ def parse_arguments():
formatter_class=ArgumentDefaultsHelpFormatter)
parser.add_argument('-s', '--bins_stat', required=True, help="Bins stat file.")
- parser.add_argument('-q', '--quast_report', required=True, help="Bins stat file.")
+ parser.add_argument('-q', '--quast_report', required=True, help="Quast report.")
parser.add_argument('-o', '--output_file', type=str,
default="bins_stat_and_quality.tsv",
@@ -90,8 +90,8 @@ def main():
df.loc['Not_binned', "Quality"] = 'Not-binned'
df.loc['Not_binned', "genome"] = 'Not_binned'
- # remove checkm2 specific columns
- df = df.drop(['Completeness_Model_Used', 'Additional_Notes'], axis=1)
+ # # remove checkm2 specific columns
+ # df = df.drop(['Completeness_Model_Used', 'Additional_Notes'], axis=1)
# Writting out table
df.to_csv(args.output_file, sep="\t", index=False)
diff --git a/bin/bin_refinement.sh b/bin/bin_refinement.sh
deleted file mode 100755
index 66eb7a5..0000000
--- a/bin/bin_refinement.sh
+++ /dev/null
@@ -1,549 +0,0 @@
-#!/usr/bin/env bash
-
-##############################################################################################################################################################
-#
-# This script is a reimplementation of metaWRAP bin_refinement script.
-#
-# We thank the author of the orginal metawrap: German Uritskiy.
-# Check metaWRAP github: https://github.com/bxlab/metaWRAP
-#
-##############################################################################################################################################################
-
-
-help_message () {
- echo ""
- echo "Usage: metaWRAP bin_refinement [options] -o output_dir -A bin_folderA [-B bin_folderB -C bin_folderC]"
- echo "Note: the contig names in different bin folders must be consistant (must come from the same assembly)."
- echo ""
- echo "Options:"
- echo ""
- echo " -o STR output directory"
- echo " -t INT number of threads (default=1)"
- echo " -m INT memory available (default=40)"
- echo " -c INT minimum % completion of bins. (default=50)"
- echo " -x INT maximum % contamination of bins that is acceptable (default=10)"
- echo ""
- echo " -A STR folder with metagenomic bins (files must have .fa or .fasta extension)"
- echo " -B STR another folder with metagenomic bins"
- echo " -C STR another folder with metagenomic bins"
- echo ""
- echo " --skip-refinement dont use binning_refiner to come up with refined bins based on combinations of binner outputs"
- echo " --skip-checkm dont run CheckM to assess bins"
- echo " --skip-consolidation choose the best version of each bin from all bin refinement iteration"
- echo " --keep-ambiguous for contigs that end up in more than one bin, keep them in all bins (default: keeps them only in the best bin)"
- echo " --remove-ambiguous for contigs that end up in more than one bin, remove them in all bins (default: keeps them only in the best bin)"
- echo "";}
-
-comm () { ${SOFT}/print_comment.py "$1" "-"; }
-error () { ${SOFT}/print_comment.py "$1" "*"; exit 1; }
-warning () { ${SOFT}/print_comment.py "$1" "*"; }
-announcement () { ${SOFT}/print_comment.py "$1" "#"; }
-
-# makes checkm plot on a folder of bins if run_checkm has already been run
-plot_checkm () {
- comm "Making CheckM plot of $1 bins"
- checkm bin_qa_plot -x fa ${1}.checkm $1 ${1}.plot
- if [[ ! -s ${1}.plot/bin_qa_plot.png ]]; then warning "Something went wrong with making the CheckM plot. Exiting."; fi
- mv ${1}.plot/bin_qa_plot.png ${1}.png
- rm -r ${1}.plot
-}
-
-
-########################################################################################################
-######################## LOADING IN THE PARAMETERS ########################
-########################################################################################################
-
-
-# setting scripts and databases from config file (should be in same folder as main script)
-# config_file=$(which config-metawrap)
-# source $config_file
-SCRIPT_DIR=$( cd -- "$( dirname -- "${BASH_SOURCE[0]}" )" &> /dev/null && pwd )
-SOFT=$SCRIPT_DIR/metawrap/
-# mw_path=$(which bin_refinement.sh)
-# SOFT=${mw_path%/*}
-
-# default params
-threads=1; mem=40; out="false"; comp=70; cont=10; x=10; c=70;
-bins1=None; bins2=None; bins3=None
-# long options defaults
-run_checkm=true; refine=true; cherry_pick=true; dereplicate=partial
-
-# load in params
-OPTS=`getopt -o ht:m:o:x:c:A:B:C: --long help,skip-checkm,skip-refinement,skip-consolidation,keep-ambiguous,remove-ambiguous -- "$@"`
-# make sure the params are entered correctly
-if [ $? -ne 0 ]; then help_message; exit 1; fi
-
-# loop through input params
-while true; do
- case "$1" in
- -t) threads=$2; shift 2;;
- -m) mem=$2; shift 2;;
- -o) out=$2; shift 2;;
- -x) cont=$2; shift 2;;
- -c) comp=$2; shift 2;;
- -A) bins1=$2; shift 2;;
- -B) bins2=$2; shift 2;;
- -C) bins3=$2; shift 2;;
- -h | --help) help_message; exit 0; shift 1;;
- --skip-checkm) run_checkm=false; shift 1;;
- --skip-refinement) refine=false; shift 1;;
- --skip-consolidation) cherry_pick=false; shift 1;;
- --keep-ambiguous) dereplicate=false; shift 1;;
- --remove-ambiguous) dereplicate=complete; shift 1;;
- --) help_message; exit 1; shift; break ;;
- *) break;;
- esac
-done
-
-########################################################################################################
-######################## MAKING SURE EVERYTHING IS SET UP ########################
-########################################################################################################
-
-# check if all parameters are entered
-if [[ $out == false ]] || [[ $bins1 == false ]] ; then
- comm "Non-optional parameters -o and/or -A were not entered"
- help_message; exit 1
-fi
-
-# Checks SOFT variable
-if [ ! -d $SOFT ]; then
- error "The folder $SOFT doesnt exist."
-fi
-
-# determine --pplacer_threads count. It is either the max thread count or RAM/40, whichever is higher
-ram_max=$(($mem / 40))
-if (( $ram_max < $threads )); then
- p_threads=$ram_max
-else
- p_threads=$threads
-fi
-
-comm "There is $mem RAM and $threads threads available, and each pplacer thread uses >40GB, so I will use $p_threads threads for pplacer"
-
-
-########################################################################################################
-######################## BEGIN REFINEMENT PIPELINE! ########################
-########################################################################################################
-announcement "BEGIN PIPELINE!"
-comm "setting up output folder and copying over bins..."
-if [[ ! -d $out ]]; then
- mkdir $out
- if [[ ! -d $out ]]; then error "cannot make $out"; fi
-else
- warning "Warning: $out already exists. Attempting to clean."
- rm -r ${out}/binsA
- rm -r ${out}/binsB
- rm -r ${out}/binsC
- rm -r ${out}/binsAB
- rm -r ${out}/binsBC
- rm -r ${out}/binsAC
- rm -r ${out}/binsABC
- rm ${out}/bin.*
-fi
-
-
-n_binnings=0
-if [[ -d $bins1 ]]; then
- mkdir ${out}/binsA
- for F in ${bins1}/*; do
- SIZE=$(stat -c%s "$F")
- if (( $SIZE > 50000)) && (( $SIZE < 20000000)); then
- BASE=${F##*/}
- cp $F ${out}/binsA/${BASE%.*}.fa
- else
- echo "Skipping $F because the bin size is not between 50kb and 20Mb"
- fi
- done
- n_binnings=$((n_binnings +1))
- comm "there are $(ls ${out}/binsA | wc -l) bins in binsA"
- if [[ $(ls ${out}/binsA | wc -l) -eq 0 ]]; then error "Please provide valid input. Exiting..."; fi
-else
- error "$bins1 is not a valid directory. Exiting."
-fi
-
-if [[ -d $bins2 ]]; then
- mkdir ${out}/binsB
- for F in ${bins2}/*; do
- SIZE=$(stat -c%s "$F")
- if (( $SIZE > 50000)) && (( $SIZE < 20000000)); then
- BASE=${F##*/}
- cp $F ${out}/binsB/${BASE%.*}.fa
- else
- echo "Skipping $F because the bin size is not between 50kb and 20Mb"
- fi
- done
- n_binnings=$((n_binnings +1))
- comm "there are $(ls ${out}/binsB | wc -l) bins in binsB"
- if [[ $(ls ${out}/binsB | wc -l) -eq 0 ]]; then error "Please provide valid input. Exiting..."; fi
-fi
-
-if [[ -d $bins3 ]]; then
- mkdir ${out}/binsC
- for F in ${bins3}/*; do
- SIZE=$(stat -c%s "$F")
- if (( $SIZE > 50000)) && (( $SIZE < 20000000)); then
- BASE=${F##*/}
- cp $F ${out}/binsC/${BASE%.*}.fa
- else
- echo "Skipping $F because the bin size is not between 50kb and 20Mb"
- fi
- done
- n_binnings=$((n_binnings +1))
- comm "there are $(ls ${out}/binsC | wc -l) bins in binsC"
- if [[ $(ls ${out}/binsC | wc -l) -eq 0 ]]; then error "Please provide valid input. Exiting..."; fi
-fi
-
-comm "There are $n_binnings bin sets!"
-
-comm "Get all contig length..."
-${SOFT}/get_contig_length.py --seq_dirs ${out}/bins* -v -x 'fa' -o ${out}/contig_to_length.tsv
-
-
-# I have to switch directories here - Binning_refiner dumps everything into the current dir"
-home=$(pwd)
-cd $out
-
-if [ "$refine" == "true" ] && [[ ! -s work_files/binsA.stats ]]; then
- announcement "BEGIN BIN REFINEMENT"
- if [[ $n_binnings -eq 1 ]]; then
- comm "There is only one bin folder, so no refinement of bins possible. Moving on..."
- elif [[ $n_binnings -eq 2 ]]; then
- comm "There are two bin folders, so we can consolidate them into a third, more refined bin set."
- ${SOFT}/binning_refiner.py -i binsA binsB -o Refined_AB
- comm "there are $(ls Refined_AB/refined_bins | grep ".fa" | wc -l) refined bins in binsAB"
- mv Refined_AB/refined_bins binsAB
- if [[ $? -ne 0 ]]; then error "Bin_refiner did not finish correctly. Exiting..."; fi
- rm -r Refined_AB
- elif [[ $n_binnings -eq 3 ]]; then
- comm "There are three bin folders, so there 4 ways we can refine the bins (A+B, B+C, A+C, A+B+C). Will try all four in parallel!"
-
- ${SOFT}/binning_refiner.py -i binsA binsB binsC -o Refined_ABC &
- ${SOFT}/binning_refiner.py -i binsA binsB -o Refined_AB &
- ${SOFT}/binning_refiner.py -i binsB binsC -o Refined_BC &
- ${SOFT}/binning_refiner.py -i binsA binsC -o Refined_AC &
-
- wait
-
- comm "there are $(ls Refined_AB/refined_bins | grep ".fa" | wc -l) refined bins in binsAB"
- comm "there are $(ls Refined_BC/refined_bins | grep ".fa" | wc -l) refined bins in binsBC"
- comm "there are $(ls Refined_AC/refined_bins | grep ".fa" | wc -l) refined bins in binsAC"
- comm "there are $(ls Refined_ABC/refined_bins | grep ".fa" | wc -l) refined bins in binsABC"
-
-
-
- mv Refined_ABC/refined_bins binsABC
- if [[ $? -ne 0 ]]; then error "Bin_refiner did not finish correctly with A+B+C. Exiting..."; fi
- rm -r Refined_ABC
-
- mv Refined_AB/refined_bins binsAB
- if [[ $? -ne 0 ]]; then error "Bin_refiner did not finish correctly with A+B. Exiting..."; fi
- rm -r Refined_AB
-
- mv Refined_BC/refined_bins binsBC
- if [[ $? -ne 0 ]]; then error "Bin_refiner did not finish correctly with B+C. Exiting..."; fi
- rm -r Refined_BC
-
- mv Refined_AC/refined_bins binsAC
- if [[ $? -ne 0 ]]; then error "Bin_refiner did not finish correctly with A+C. Exiting..."; fi
- rm -r Refined_AC
- else
- error "Something is off here - somehow there are not 1, 2, or 3 bin folders ($n_binnings)"
- fi
- comm "Bin refinement finished successfully!"
-elif [ "$refine" == "true" ] && [[ -s work_files/binsM.stats ]]; then
- comm "Previous bin refinment files found. If this was not intended, please re-run with a clear output directory. Skipping refinement..."
-else
- comm "Skipping bin refinement. Will proceed with the $n_binnings bins specified."
-fi
-
-comm "fixing bin naming to .fa convention for consistancy..."
-for i in $(ls); do
- for j in $(ls $i | grep .fasta); do
- mv ${i}/${j} ${i}/${j%.*}.fa
- done
-done
-
-comm "making sure every refined bin set contains bins..."
-for bin_set in $(ls | grep bins); do
- if [[ $(ls $bin_set|grep -c fa) == 0 ]]; then
- comm "Removing bin set $bin_set because it yielded 0 refined bins ... "
- rm -r $bin_set
- fi
-done
-
-
-########################################################################################################
-######################## RUN CHECKM ON ALL BIN SETS ########################
-########################################################################################################
-if [ "$run_checkm" == "true" ] && [[ ! -s work_files/binsM.stats ]]; then
- announcement "RUNNING CHECKM ON ALL SETS OF BINS"
-
-
- out_checkM2_tmp="checkm2_tmp_results/"
- mkdir -p $out_checkM2_tmp
- comm "Dereplicate and concat all contigs."
- bin_sets=$(ls | grep -v tmp | grep -v stats | grep bins)
- ${SOFT}/dereplicate_contigs.py -v \
- --seq_dirs $bin_sets \
- -o ${out_checkM2_tmp}/all_contigs.fna
-
-
-
- comm "Launching pprodigal on all contigs"
-
- # cp ../all_contigs_genes.faa ${out_checkM2_tmp}/all_contigs_genes.faa
- time pprodigal -T $threads -p meta \
- -m -f gff \
- -g 11 -a ${out_checkM2_tmp}/all_contigs_genes.faa \
- -d ${out_checkM2_tmp}/all_contigs_genes.fna \
- -i ${out_checkM2_tmp}/all_contigs.fna > ${out_checkM2_tmp}/all_contigs_genes.gff
-
-
- comm "Launching DIAMOND on all proteins"
-
- checkm2_database=$(checkm2 database --current 2>&1 | rev | cut -f1 -d' ' | rev)
-
- cp ../DIAMOND_RESULTS.tsv ${out_checkM2_tmp}/DIAMOND_RESULTS.tsv
- time diamond blastp --outfmt 6 --max-target-seqs 1 \
- --query ${out_checkM2_tmp}/all_contigs_genes.faa \
- -o ${out_checkM2_tmp}/DIAMOND_RESULTS.tsv \
- --threads $threads --db $checkm2_database \
- --query-cover 80 --subject-cover 80 --id 30 \
- --evalue 1e-05 --block-size 0.5
-
-
- for bin_set in $(ls | grep -v tmp | grep -v stats | grep bins); do
-
-
- if [[ -d ${bin_set}.checkm ]]; then rm -r ${bin_set}.checkm; fi
- if [[ ! -d ${bin_set}.tmp ]]; then mkdir ${bin_set}.tmp; fi
-
- comm "Setting up checkm2 output to use resume flag."
-
- python ${SOFT}/setup_indermediate_checkm2_files.py -b $bin_set \
- -d ${out_checkM2_tmp}/DIAMOND_RESULTS.tsv \
- -f ${out_checkM2_tmp}/all_contigs_genes.faa \
- -o ${bin_set}.checkm -v
-
- comm "Running CheckM on $bin_set bins with resume flag"
-
- checkm2 predict -x fa --threads $threads --input $bin_set --output-directory ${bin_set}.checkm --resume
-
-
- if [[ ! -s ${bin_set}.checkm/quality_report.tsv ]]; then error "Something went wrong with running CheckM. Exiting..."; fi
-
- ${SOFT}/add_metrics_to_checkm.py \
- --bin_dir ${bin_set} \
- --contig_to_length_tsv contig_to_length.tsv \
- --checkm2_result ${bin_set}.checkm/quality_report.tsv \
- -o ${bin_set}.stats
-
- if [[ $? -ne 0 ]];
- then
- error "Cannot add metrics checkm summary file. Exiting.";
- fi
-
- rm -r ${bin_set}.checkm; rm -r ${bin_set}.tmp
-
- num=$(cat ${bin_set}.stats | awk -v c="$comp" -v x="$cont" '{if ($2>=c && $2<=100 && $3>=0 && $3<=x) print $1 }' | wc -l)
- comm "There are $num 'good' bins found in $bin_set! (>${comp}% completion and <${cont}% contamination)"
- done
- # cp ../backup_stats/* .
-elif [ "$run_checkm" == "true" ] && [[ -s work_files/binsM.stats ]]; then
- comm "Previous bin refinement files found. If this was not intended, please re-run with a clear output directory. Skipping CheckM runs..."
- rm -r bins*
- cp -r work_files/binsA* ./
- cp -r work_files/binsB* ./
- cp -r work_files/binsC* ./
-else
- comm "Skipping CheckM. Warning: bin consolidation will not be possible."
-fi
-
-########################################################################################################
-######################## CONSOLIDATE ALL BIN SETS ########################
-########################################################################################################
-if [ "$cherry_pick" == "true" ]; then
- announcement "CONSOLIDATING ALL BIN SETS BY CHOOSING THE BEST VERSION OF EACH BIN"
- if [[ $n_binnings -eq 1 ]]; then
- comm "There is only one original bin folder, so no refinement of bins possible. Moving on..."
- best_bin_set=binsA
- elif [[ $n_binnings -eq 2 ]] || [[ $n_binnings -eq 3 ]]; then
- comm "There are $n_binnings original bin folders, plus the refined bins."
- rm -r binsM binsM.stats
-
- stats=$(ls | grep .stats | grep -v binsM)
- bins=$(ls | grep bins | grep -v .stats | grep -v binsM)
-
- comm "merging all $bins"
- echo $bins $stats
- ${SOFT}/consolidate_multiple_bins_sets.py --bin_dirs $bins --bin_stats $stats \
- -l contig_to_length.tsv -v -o binsM \
- --min_completeness $comp --max_contamination $cont
-
-
- exitcode=$?
-
- if [[ exitcode -ne 0 ]]; then
- comm "Something went wrong with merging two sets of bins"
- exit $exitcode;
-
- fi
-
- if [[ $dereplicate == false ]]; then
- comm "Skipping dereplication of contigs between bins..."
- mv binsM binsO
- mv binsM.stats binsO.stats
- elif [[ $dereplicate == partial ]]; then
- comm "Scanning to find duplicate contigs between bins and only keep them in the best bin..."
- ${SOFT}/dereplicate_contigs_in_bins.py --bins_stat binsM.stats --bin_dir binsM --out_bin_dir binsO -v
- elif [[ $dereplicate == complete ]]; then
- comm "Scanning to find duplicate contigs between bins and deleting them in all bins..."
- ${SOFT}/dereplicate_contigs_in_bins.py --bins_stat binsM.stats --bin_dir binsM --out_bin_dir binsO -v --remove
- else
- error "there was an error in deciding how to dereplicate contigs"
- fi
-
- best_bin_set=binsO
- else
- error "Something went wrong with determining the number of bin folders... The number was ${n_binnings}. Exiting."
- fi
-
-
-elif [ "$cherry_pick" == "false" ]; then
- comm "Skipping bin consolidation. Will try to pick the best binning folder without mixing bins from different sources."
- if [ $run_checkm = false ]; then
- comm "cannot decide on best bin set because CheckM was not run. Will assume its binsA (first bin set)"
- best_bin_set=binsA
- elif [ $run_checkm = true ]; then
- max=0
- best_bin_set=none
- for bin_set in $(ls | grep .stats); do
- num=$(cat $bin_set | awk -v c="$comp" -v x="$cont" '{if ($2>=c && $2<=100 && $3>=0 && $3<=x) print $1 }' | wc -l)
- comm "There are $num 'good' bins found in ${bin_set%.*}! (>${comp}% completion and <${cont}% contamination)"
- if [ "$num" -gt "$max" ]; then
- max=$num
- best_bin_set=${bin_set%.*}
- fi
- done
- if [[ ! -d $best_bin_set ]]; then error "Something went wrong with deciding on the best bin set. Exiting."; fi
- comm "looks like the best bin set is $best_bin_set"
- else
- error "something is wrong with the run_checkm option (${run_checkm})"
- fi
-else
- error "something is wrong with the cherry_pick option (${cherry_pick})"
-fi
-
-comm "You will find the best non-reassembled versions of the bins in $best_bin_set"
-
-
-########################################################################################################
-######################## FINALIZING THE REFINED BINS ########################
-########################################################################################################
-announcement "FINALIZING THE REFINED BINS"
-
-
-if [ "$run_checkm" == "true" ] && [ $dereplicate != "false" ]; then
-
- mkdir binsO.tmp
- comm "Setting up checkm2 output to use resume flag on binsO."
-
- python ${SOFT}/setup_indermediate_checkm2_files.py -b binsO \
- -d ${out_checkM2_tmp}/DIAMOND_RESULTS.tsv \
- -f ${out_checkM2_tmp}/all_contigs_genes.faa \
- -o binsO.checkm -v
-
- comm "Re-running CheckM on binsO bins"
-
- checkm2 predict -x fa --threads $threads --input binsO --output-directory binsO.checkm --resume
-
-
- if [[ ! -s binsO.checkm/quality_report.tsv ]]; then error "Something went wrong with running CheckM. Exiting..."; fi
- rm -r binsO.tmp
-
- ${SOFT}/add_metrics_to_checkm.py \
- --bin_dir binsO \
- --contig_to_length_tsv contig_to_length.tsv \
- --checkm2_result binsO.checkm/quality_report.tsv -o binsO.stats
-
- if [[ $? -ne 0 ]]; then error "Cannot make checkm summary file. Exiting."; fi
- rm -r binsO.checkm
- num=$(cat binsO.stats | awk -v c="$comp" -v x="$cont" '{if ($2>=c && $2<=100 && $3>=0 && $3<=x) print $1 }' | wc -l)
- comm "There are $num 'good' bins found in binsO.checkm! (>${comp}% completion and <${cont}% contamination)"
-
- comm "Removing bins that are inadequate quality..."
- for bin_name in $(cat binsO.stats | grep -v compl | awk -v c="$comp" -v x="$cont" '{if ($2<c || $2>100 || $3<0 || $3>x) print $1 }' | cut -f1); do
- echo "${bin_name} will be removed because it fell below the quality threshhold after de-replication of contigs..."
- rm binsO/${bin_name}.fa
- done
- head -n 1 binsO.stats > binsO.stats.tmp
- cat binsO.stats | awk -v c="$comp" -v x="$cont" '$2>=c && $2<=100 && $3>=0 && $3<=x' >> binsO.stats.tmp
- mv binsO.stats.tmp binsO.stats
- n=$(cat binsO.stats | grep -v comp | wc -l)
- comm "Re-evaluating bin quality after contig de-replication is complete! There are still $n high quality bins."
-fi
-
-
-if [ "$run_checkm" == "true" ]; then
- comm "making completion and contamination ranking plots for all refinement iterations"
- ${SOFT}/plot_binning_results.py $comp $cont $(ls | grep ".stats")
- mkdir figures
- mv binning_results.png figures/intermediate_binning_results.png
-fi
-
-########################################################################################################
-######################## MOVING OVER TEMPORARY FILES ########################
-########################################################################################################
-announcement "MOVING OVER TEMPORARY FILES"
-
-if [ "${bins1:$((${#bins1}-1)):1}" = "/" ]; then bins1=${bins1%/*}; fi
-if [ "${bins2:$((${#bins2}-1)):1}" = "/" ]; then bins2=${bins2%/*}; fi
-if [ "${bins3:$((${#bins3}-1)):1}" = "/" ]; then bins3=${bins3%/*}; fi
-
-
-if [[ -s work_files/binsM.stats ]]; then
- rm -r work_files/bins*
- rm -r ${bins1##*/}* ${bins2##*/}* ${bins3##*/}*
-fi
-
-if [[ $n_binnings -ne 1 ]]; then
- mkdir work_files
- for f in binsA* binsB* binsC* binsM* binsO*; do
- mv $f work_files/
- done
-fi
-
-
-cp -r work_files/binsO metawrap_${comp}_${cont}_bins
-cp work_files/binsO.stats metawrap_${comp}_${cont}_bins.stats
-
-cp -r work_files/binsA ${bins1##*/}
-cp work_files/binsA.stats ${bins1##*/}.stats
-
-cp -r work_files/binsB ${bins2##*/}
-cp work_files/binsB.stats ${bins2##*/}.stats
-
-if [[ $n_binnings -eq 3 ]]; then
- cp -r work_files/binsC ${bins3##*/}
- cp work_files/binsC.stats ${bins3##*/}.stats
-fi
-
-
-if [ "$run_checkm" == "true" ]; then
- comm "making completion and contamination ranking plots of final outputs"
- ${SOFT}/plot_binning_results.py $comp $cont $(ls | grep ".stats")
- mv binning_results.png figures/binning_results.png
-
- comm "making contig membership files (for Anvio and other applications)"
- for dir in *_bins; do
- echo "summarizing $dir ..."
- for i in ${dir}/*.fa; do f=${i##*/}; for c in $(cat $i | grep ">"); do echo -e "${c##*>}\t${f%.*}"; done; done > ${dir}.contigs
- done
-fi
-
-cd $home
-
-########################################################################################################
-######################## BIN_REFINEMENT PIPELINE SUCCESSFULLY FINISHED!!! ########################
-########################################################################################################
-announcement "BIN_REFINEMENT PIPELINE FINISHED SUCCESSFULLY!"
\ No newline at end of file
diff --git a/bin/metawrap/add_metrics_to_checkm.py b/bin/metawrap/add_metrics_to_checkm.py
deleted file mode 100755
index ca4e8fa..0000000
--- a/bin/metawrap/add_metrics_to_checkm.py
+++ /dev/null
@@ -1,134 +0,0 @@
-#!/usr/bin/env python3
-
-"""
-Add N50 and size of bins to checkm2 results.
-
-:Example:
-add_metrics_to_checkm.py -h
-"""
-
-# Metadata
-__author__ = 'Mainguy Jean - Plateforme bioinformatique Toulouse'
-__copyright__ = 'Copyright (C) 2022 INRAE'
-__license__ = 'GNU General Public License'
-__version__ = '0.1'
-__email__ = 'support.bioinfo.genotoul@inra.fr'
-__status__ = 'dev'
-
-
-from argparse import ArgumentParser, ArgumentDefaultsHelpFormatter
-import logging
-from Bio import SeqIO
-import os
-import pandas as pd
-
-
-def parse_arguments():
- """Parse script arguments."""
- parser = ArgumentParser(description="...",
- formatter_class=ArgumentDefaultsHelpFormatter)
-
- parser.add_argument("-r", '--checkm2_result', required=True, help="result file made by checkm2.")
- parser.add_argument("-l", '--contig_to_length_tsv', required=True, help="Contig to length file.")
- parser.add_argument("-b", '--bin_dir', required=True, help="Bin set directory.")
-
- parser.add_argument("-o", "--outfile", default='checkm2_result_with_metrics.tsv' )
- parser.add_argument('-x', '--extension', help='Extension of input files. [Default: .fa]', default='.fa')
-
- parser.add_argument("-v", "--verbose", help="increase output verbosity",
- action="store_true")
-
- args = parser.parse_args()
- return args
-
-
-def compute_N50(list_of_lengths):
- """Calculate N50 for a sequence of numbers.
-
- Args:
- list_of_lengths (list): List of numbers.
-
- Returns:
- int: N50 value.
-
- """
-
- list_of_lengths = sorted(list_of_lengths)
-
- sum_len = sum(list_of_lengths)
-
- cum_length = 0
- for length in list_of_lengths:
- if cum_length + length >= sum_len/2:
- return length
- cum_length += length
- return length
-
-
-def get_bin_composition(fasta_bins):
-
- bin2contigs = {}
-
- for seq_file in fasta_bins:
- bin2contigs['.'.join(os.path.basename(seq_file).split('.')[:-1])] = [record.id for record in SeqIO.parse(seq_file, "fasta")]
-
- return bin2contigs
-
-
-def get_bin_N50(bin_id, bin2contigs, df_len):
- contigs = bin2contigs[(str(bin_id))]
- lengths = df_len.loc[contigs, "length"]
- return compute_N50(lengths)
-
-
-def get_bin_size(bin_id, bin2contigs, df_len):
- contigs = bin2contigs[(str(bin_id))]
- return df_len.loc[contigs, "length"].sum()
-
-
-def main():
-
- args = parse_arguments()
-
- if args.verbose:
- logging.basicConfig(format="%(levelname)s: %(message)s", level=logging.DEBUG)
- logging.info('Mode verbose ON')
-
- else:
- logging.basicConfig(format="%(levelname)s: %(message)s")
-
-
- contig_to_length_tsv = args.contig_to_length_tsv
- bin_dir = args.bin_dir
- fasta_extension = args.extension
-
- checkm2_result = args.checkm2_result
-
- logging.info(f"Parsing {contig_to_length_tsv}.")
-
- df_len = pd.read_csv(contig_to_length_tsv, sep="\t", index_col="seqid")
-
- logging.info("Get bin composition by parsing bin fasta files.")
-
- bin_files = [os.path.join(bin_dir, seq_file) for seq_file in os.listdir(bin_dir) if seq_file.endswith(fasta_extension)]
-
- bin2contigs = get_bin_composition(bin_files)
-
- logging.info("Parse checkM2 results")
- df_checkm = pd.read_csv(checkm2_result, sep="\t")
-
- df_checkm["N50"] = df_checkm["Name"].apply(
- lambda x: get_bin_N50(x, bin2contigs, df_len)
- )
- df_checkm["Size"] = df_checkm["Name"].apply(
- lambda x: get_bin_size(x, bin2contigs, df_len)
- )
-
- # metawrap identifies header with completeness in lower case
- df_checkm = df_checkm.rename(columns={"Name":"genome", "Completeness": "completeness", "Contamination": "contamination"})
-
- df_checkm.to_csv(args.outfile, sep="\t", index=False)
-
-
-if __name__ == "__main__":
- main()
diff --git a/bin/metawrap/binning_refiner.py b/bin/metawrap/binning_refiner.py
deleted file mode 100755
index fdf255f..0000000
--- a/bin/metawrap/binning_refiner.py
+++ /dev/null
@@ -1,269 +0,0 @@
-#!/usr/bin/env python3
-
-"""
-This script version 1.4.0 (2019-04-19) has been taken from https://github.com/songweizhi/Binning_refiner.
-
-"""
-
-
-# Copyright (C) 2017, Weizhi Song, Torsten Thomas.
-# songwz03@gmail.com or t.thomas@unsw.edu.au
-
-# Binning_refiner is free software: you can redistribute it and/or modify
-# it under the terms of the GNU Affero General Public License as published by
-# the Free Software Foundation, either version 3 of the License, or
-# (at your option) any later version.
-
-# Binning_refiner is distributed in the hope that it will be useful,
-# but WITHOUT ANY WARRANTY; without even the implied warranty of
-# MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the
-# GNU Affero General Public License for more details.
-
-# You should have received a copy of the GNU Affero General Public License
-# along with this program. If not, see <http://www.gnu.org/licenses/>.
-
-import os
-import shutil
-import argparse
-from Bio import SeqIO
-from datetime import datetime
-import sys
-
-
-def force_create_folder(folder_to_create):
- if os.path.isdir(folder_to_create):
- shutil.rmtree(folder_to_create, ignore_errors=True)
- if os.path.isdir(folder_to_create):
- shutil.rmtree(folder_to_create, ignore_errors=True)
- if os.path.isdir(folder_to_create):
- shutil.rmtree(folder_to_create, ignore_errors=True)
- if os.path.isdir(folder_to_create):
- shutil.rmtree(folder_to_create, ignore_errors=True)
- os.mkdir(folder_to_create)
-
-
-def get_no_hidden_folder_list(wd):
-
- folder_list = []
- for each_folder in os.listdir(wd):
- if not each_folder.startswith('.'):
- folder_list.append(each_folder)
-
- folder_list_sorte = sorted(folder_list)
-
- return folder_list_sorte
-
-
-######################################## CONFIGURATION #######################################
-
-parser = argparse.ArgumentParser()
-
-parser.add_argument('-i', required=True, nargs='+', help='input bin folders')
-parser.add_argument('-o', required=False, default='Refined_results', help='output directory, default: Refined_results')
-parser.add_argument('-m', required=False, default=512, type=int, help='minimal size (Kbp) of refined bin, default: 512')
-parser.add_argument("-s", help="Write stats files on refined bins.", action="store_true")
-parser.add_argument('-q', required=False, default=False, help='silent progress report')
-
-args = vars(parser.parse_args())
-
-pwd_bin_subfolders = args['i']
-output_dir = args['o']
-minBin_size_Kbp = args['m']
-keep_quiet = args['q']
-write_stat_files = args['s']
-
-################################### define output filename ###################################
-
-pwd_report_file_sources_and_length = os.path.join(output_dir, 'sources_and_length.txt')
-pwd_report_file_contigs = os.path.join(output_dir, 'contigs.txt')
-pwd_report_file_for_sankey = os.path.join(output_dir, 'sankey.txt')
-pwd_output_bin_folder = os.path.join(output_dir, 'refined_bins')
-
-force_create_folder(output_dir)
-force_create_folder(pwd_output_bin_folder)
-
-time_format = '[%Y-%m-%d %H:%M:%S]'
-
-
-################################### precheck of input files ##################################
-
-for pwd_bin_subfolder in pwd_bin_subfolders:
-
- bin_subfolder = os.path.basename(pwd_bin_subfolder)
-
- # get bin file list in each input bin subfolder
- bin_file_list = get_no_hidden_folder_list(pwd_bin_subfolder)
-
- bin_ext_list = set()
- for each_bin in bin_file_list:
- each_bin_ext = each_bin.split('.')[-1]
- bin_ext_list.add(each_bin_ext)
-
- if len(bin_ext_list) > 1:
- print('Program exited, please make sure all bins within %s folder have the same extension' % bin_subfolder, file=sys.stderr)
- exit(1)
-
-
-##################################### refine input bins ######################################
-
-# progress report
-if keep_quiet is False:
- print('%s Processing %s input bin folders' % ((datetime.now().strftime(time_format)), len(pwd_bin_subfolders)))
-
-ctg_length_dict = {}
-ctg_to_bin_dict = {}
-for pwd_bin_subfolder in pwd_bin_subfolders:
-
- bin_subfolder = os.path.basename(pwd_bin_subfolder)
-
- # get bin file list in each input bin subfolder
- bin_file_list = get_no_hidden_folder_list(pwd_bin_subfolder)
-
-
- # progress report
- if keep_quiet is False:
- print('%s Read in %s %s bins' % ((datetime.now().strftime(time_format)), len(bin_file_list), bin_subfolder))
-
-
- for each_bin in bin_file_list:
- pwd_each_bin = os.path.join(pwd_bin_subfolder, each_bin)
-
- for each_seq in SeqIO.parse(pwd_each_bin, 'fasta'):
- each_seq_id = str(each_seq.id)
- each_seq_len = len(each_seq.seq)
-
- # store contig length into dict
- if each_seq_id not in ctg_length_dict:
- ctg_length_dict[each_seq_id] = each_seq_len
-
- # store contig to bin info into dict
- if each_seq_id not in ctg_to_bin_dict:
- ctg_to_bin_dict[each_seq_id] = [each_bin]
- else:
- ctg_to_bin_dict[each_seq_id].append(each_bin)
-
-
-# progress report
-if keep_quiet is False:
- print('%s Refine input bins' % (datetime.now().strftime(time_format)))
-
-
-# only keep contigs existed in all bin sets
-ctg_to_bin_dict_shared = {}
-for each_ctg in ctg_to_bin_dict:
- if len(ctg_to_bin_dict[each_ctg]) == len(pwd_bin_subfolders):
- ctg_to_bin_dict_shared[each_ctg] = '___'.join(ctg_to_bin_dict[each_ctg])
-
-
-# get concatenated_bins_to_ctg_dict
-concatenated_bins_to_ctg_dict = {}
-concatenated_bins_length_dict = {}
-for each_shared_ctg in ctg_to_bin_dict_shared:
-
- concatenated_bins = ctg_to_bin_dict_shared[each_shared_ctg]
-
- if concatenated_bins not in concatenated_bins_to_ctg_dict:
- concatenated_bins_to_ctg_dict[concatenated_bins] = [each_shared_ctg]
- concatenated_bins_length_dict[concatenated_bins] = ctg_length_dict[each_shared_ctg]
- else:
- concatenated_bins_to_ctg_dict[concatenated_bins].append(each_shared_ctg)
- concatenated_bins_length_dict[concatenated_bins] += ctg_length_dict[each_shared_ctg]
-
-
-# remove short length
-concatenated_bins_to_ctg_dict_short_removed = {}
-concatenated_bins_length_dict_short_removed = {}
-maximum_length = 0
-for concatenated_bins in concatenated_bins_length_dict:
- concatenated_bins_length = concatenated_bins_length_dict[concatenated_bins]
- if concatenated_bins_length >= minBin_size_Kbp * 1024:
- concatenated_bins_to_ctg_dict_short_removed[concatenated_bins] = concatenated_bins_to_ctg_dict[concatenated_bins]
- concatenated_bins_length_dict_short_removed[concatenated_bins] = concatenated_bins_length_dict[concatenated_bins]
-
- if concatenated_bins_length_dict[concatenated_bins] > maximum_length:
- maximum_length = concatenated_bins_length_dict[concatenated_bins]
-
-
-# add leading zero to length
-concatenated_bins_length_dict_short_removed_str = {}
-for each_length in concatenated_bins_length_dict_short_removed:
- concatenated_bins_length_dict_short_removed_str[each_length] = ("{:0%sd}" % len(str(maximum_length))).format(concatenated_bins_length_dict_short_removed[each_length])
-
-
-concatenated_bins_list = []
-for each_concatenated_bin in concatenated_bins_length_dict_short_removed_str:
- concatenated_bins_list.append('%s___%s' % (concatenated_bins_length_dict_short_removed_str[each_concatenated_bin], each_concatenated_bin))
-
-
-# sort concatenated_bins
-concatenated_bins_list_sorted = sorted(concatenated_bins_list, reverse=True)
-
-
-# progress report
-if keep_quiet is False:
- print('%s Got %s refined bins with size larger than %s Kbp' % ((datetime.now().strftime(time_format)), len(concatenated_bins_list_sorted), minBin_size_Kbp))
-
-
-# progress report
-if keep_quiet is False:
- print('%s %s' % ((datetime.now().strftime(time_format)), 'Extract sequences for refined bins'))
-
-if write_stat_files:
- # extract sequences and write out report file
- pwd_report_file_sources_and_length_handle = open(pwd_report_file_sources_and_length, 'w')
- pwd_report_file_sources_and_length_handle.write('Refined_bin\tSize(Kbp)\tSource\n')
-
- pwd_report_file_contigs_handle = open(pwd_report_file_contigs, 'w')
- pwd_report_file_contigs_handle.write('Refined_bin\tContigs\n')
-
- pwd_report_file_for_sankey_handle = open(pwd_report_file_for_sankey, 'w')
- pwd_report_file_for_sankey_handle.write('C1,C2,Length_Kbp\n')
-
-refined_bin_index = 1
-for refined_bin in concatenated_bins_list_sorted:
- refined_bin_id = f"refined_bin{refined_bin_index}"
-
- refined_bin_split = refined_bin.split('___')
- pwd_ctg_source = os.path.join(pwd_bin_subfolders[0], refined_bin_split[1])
- pwd_refined_bin = os.path.join(pwd_output_bin_folder, f"{refined_bin_id}.fasta")
-
- refined_bin_ctgs = concatenated_bins_to_ctg_dict['___'.join(refined_bin_split[1:])]
-
- # extract sequences for refined bin
- with open(pwd_refined_bin, 'w') as pwd_refined_bin_handle:
- for each_source_ctg in SeqIO.parse(pwd_ctg_source, 'fasta'):
- if str(each_source_ctg.id) in refined_bin_ctgs:
- SeqIO.write(each_source_ctg, pwd_refined_bin_handle, 'fasta')
-
-
- if write_stat_files:
-
- refined_bin_source = refined_bin_split[1:]
-
- # write out sources_and_length file
- refined_bin_length = concatenated_bins_length_dict_short_removed['___'.join(refined_bin_split[1:])]
- refined_bin_length_Kbp = float("{0:.2f}".format(refined_bin_length/1024))
- pwd_report_file_sources_and_length_handle.write('%s\t%s\t%s\n' % (refined_bin_id, refined_bin_length_Kbp, ','.join(refined_bin_source)))
-
- # write out contig file
- pwd_report_file_contigs_handle.write('%s\t%s\n' % (refined_bin_id, ','.join(sorted(refined_bin_ctgs))))
-
- # write out file for sankey plot
- n = 0
- while n <= len(refined_bin_source) - 2:
- pwd_report_file_for_sankey_handle.write('%s\n' % ','.join([refined_bin_source[n], refined_bin_source[n+1], str(refined_bin_length_Kbp)]))
- n += 1
-
-
- refined_bin_index += 1
-
-
-if write_stat_files:
- pwd_report_file_sources_and_length_handle.close()
- pwd_report_file_contigs_handle.close()
- pwd_report_file_for_sankey_handle.close()
-
-
-# progress report
-if keep_quiet is False:
- print('%s %s' % ((datetime.now().strftime(time_format)), 'Done!'))
diff --git a/bin/metawrap/consolidate_multiple_bins_sets.py b/bin/metawrap/consolidate_multiple_bins_sets.py
deleted file mode 100755
index 460342c..0000000
--- a/bin/metawrap/consolidate_multiple_bins_sets.py
+++ /dev/null
@@ -1,260 +0,0 @@
-#!/usr/bin/env python3
-
-"""
-This script takes multiple folders containing bins from different methods and their metrics files generated by CheckM.
-Then start by matching corresponding bins in the two first sets based on a minimum of 80% overlap (by length) of the bins and decides which of the two
-bin versions is best in that particular bin, making a new bins set.
-This new bin set is then used in the same way with the third bins sets.
-
-Then it makes a new folder into which it puts the best version of each bin (changing the naming in
-the process), and also makes a new .stats file which is consistant with the new bin folder.
-
-:Example:
-consolidate_multiple_bins_sets.py --bin_dirs binsA binsAB binsABC --bin_stats binsABC.stats binsAB.stats binsA.stats -l contig_to_length.tsv
-"""
-
-# Metadata
-__author__ = "Mainguy Jean - Plateforme bioinformatique Toulouse"
-__copyright__ = "Copyright (C) 2022 INRAE"
-__license__ = "GNU General Public License"
-__version__ = "0.1"
-__email__ = "support.bioinfo.genotoul@inra.fr"
-__status__ = "dev"
-
-
-from argparse import ArgumentParser, ArgumentDefaultsHelpFormatter
-import logging
-import os
-import pandas as pd
-from Bio import SeqIO
-from collections import defaultdict
-import shutil
-from functools import reduce
-import sys
-pd.set_option('display.max_rows', 200)
-
-def parse_arguments():
- """Parse script arguments."""
- parser = ArgumentParser(
- description="...", formatter_class=ArgumentDefaultsHelpFormatter
- )
-
- parser.add_argument("--bin_stats", required=True, nargs="+",
- help="Stat file on bins sets.")
- parser.add_argument("--bin_dirs", required=True,
- nargs="+", help="Directories of bins sets.")
-
- parser.add_argument("-l", '--contig_to_length_tsv',
- required=True, help="Contig to length file.")
-
- parser.add_argument('-o', "--out_bin", default="consolidated_bins", help="Consolidated final bins set name. Create a directory <out_bin> and a tsv with bins stat <out_bin>.stats")
-
- parser.add_argument("--min_completeness", default=50, type=int, help="Bin minimum completeness treshold.")
-
- parser.add_argument("--max_contamination", default=10, type=int, help="Bin maximum contamination threshold.")
- parser.add_argument("--min_overlap", default=80, type=int, help="Minimum overlap percentage (by length) between two bins to consider them corresponding.")
-
- parser.add_argument(
- "-v", "--verbose", help="increase output verbosity", action="store_true"
- )
-
- args = parser.parse_args()
- return args
-
-
-def get_bin_contigs(seqfile):
- return {record.id for record in SeqIO.parse(seqfile, "fasta")}
-
-
-def get_bin_file(bin_dir, bin_id):
- bin_file = os.path.join(bin_dir, f"{bin_id}.fa")
- if not os.path.isfile(bin_file):
- raise FileNotFoundError(f"Bin file {bin_id}.fa does not exists in {bin_dir}")
- return bin_file
-
-
-def parse_bin_set_stats(bin_dir, stat_file, min_complet, max_conta):
- logging.info(f"Parsing {stat_file} and {bin_dir}")
-
- df = pd.read_csv(stat_file, sep="\t")
-
- df["genome"] = df["genome"].astype(str)
-
- df["bin_file"] = df["genome"].apply(lambda x: get_bin_file(bin_dir, x))
-
- df = df.set_index("bin_file", drop=False)
-
- # # keep only good bin.
- good_bin_filt = (df["completeness"] >= min_complet) & (
- df["contamination"] <= max_conta
- )
- df = df.loc[good_bin_filt]
-
- # score each bin
-
- df["score"] = df["completeness"] - df["contamination"] * 5
-
- # get contigs of each bin
-
- df["contigs"] = df["bin_file"].apply(lambda x: get_bin_contigs(x))
-
- df['origin'] = bin_dir
-
- return df
-
-
-def check_input_files_consistency(bin_dirs, stat_files):
- logging.info("Checking input files consistency.")
- if len(bin_dirs) != len(stat_files):
- logging.error(
- f"Number of bin dirs ({len(bin_dirs)}) and stat files ({len(stat_files)}) given to the script are different."
- )
- exit(1)
-
- bin_name2bin_dir = {
- os.path.basename(os.path.normpath(bin_dir)): bin_dir for bin_dir in bin_dirs
- }
- bin_dir_stat_file_tuples = []
-
- for stat_file in stat_files:
-
- bins_set_name = ".".join(os.path.basename(stat_file).split(".")[:-1])
- try:
- bin_dir = bin_name2bin_dir[bins_set_name]
- except KeyError:
- raise FileNotFoundError(
- f"Stat file {stat_file} has no matching bin dir. Expecting bin dir basename: {bins_set_name}"
- )
-
- if not os.path.isdir(bin_dir):
- raise FileNotFoundError(f"Bin dir does not exists: {bin_dirs}.")
-
- bin_dir_stat_file_tuples.append((bin_dir, stat_file))
-
- return bin_dir_stat_file_tuples
-
-
-def consolidate_two_bins_sets(df_bins_1, df_bins_2, df_len, min_overlap):
- df_bins_1 = df_bins_1[~df_bins_1.index.duplicated(keep='first')]
- df_bins_2 = df_bins_2[~df_bins_2.index.duplicated(keep='first')]
- #logging.info(f"consolidate {set(df_bins_1['origin'])} with {set(df_bins_2['origin'])} ")
-
- df_bins = pd.concat([df_bins_1, df_bins_2])
-
- bin_pairs, lonely_bins_2 = get_overlapping_bins(df_bins_1, df_bins_2, df_len, min_overlap )
-
- selected_bins = []
- # go through all overlapping bins and chose the best ones
- for bin_1, bins_2 in bin_pairs.items():
-
- index_bins = [bin_1] + bins_2
- if len(bins_2) > 1:
- logging.info(f"The bin {bin_1} overlap with multiple bins: {bins_2}")
-
- df_bin_group = df_bins.loc[index_bins]
-
- selected_bin = df_bin_group.loc[df_bin_group['score'].idxmax()]
- selected_bins.append(selected_bin)
-
-
- selected_bins += [df_bins.loc[bin_file] for bin_file in lonely_bins_2]
- df_select = pd.DataFrame(selected_bins)
- return df_select
-
-def get_overlapping_bins(bins_1, bins_2, df_len, min_overlap ):
-
- bin_pairs = {}
- set2_paired_bins = set()
-
- for index1, row1 in bins_1.iterrows():
- bin_pairs[index1] = []
- for index2, row2 in bins_2.iterrows():
-
- shared_contigs = row1['contigs'] & row2['contigs']
-
- if len(shared_contigs) == 0:
- continue
-
- bin1_size = df_len.loc[list(row1['contigs']), 'length'].sum()
- bin2_size = df_len.loc[list(row2['contigs']), 'length'].sum()
-
- shared_length = df_len.loc[list(shared_contigs), 'length'].sum()
-
- # chose the highest % ID, depending of which bin is a subset of the other
- bin_overlaping_prct = 100 * max([shared_length/bin1_size, shared_length/bin2_size])
-
- if bin_overlaping_prct >= min_overlap:
-
- bin_pairs[index1].append(index2)
-
- set2_paired_bins.add(index2)
-
- logging.info(f"There are {len(bins_1)} bins in bins1 and {len(bin_pairs)} of them are pairing with bins_2")
-
- logging.info(f"There are {len(bins_2)} bins in bins2 and {len(set2_paired_bins)} of them are pairing with bins1")
-
- lonely_bins_2 = set(bins_2.index) - set2_paired_bins
- return bin_pairs, lonely_bins_2
-
-def main():
-
- args = parse_arguments()
-
- if args.verbose:
- logging.basicConfig(format="%(levelname)s: %(message)s", level=logging.DEBUG)
- logging.info("Mode verbose ON")
-
- else:
- logging.basicConfig(format="%(levelname)s: %(message)s")
-
- min_complet = args.min_completeness
- max_conta = args.max_contamination
-
- min_overlap = 80
-
- contig_to_length_tsv = args.contig_to_length_tsv
-
- checkm_results = (args.bin_stats)
- bin_dirs = args.bin_dirs
-
-
- out_bin = args.out_bin
- out_bin_stats = f"{out_bin}.stats"
- os.makedirs(out_bin, exist_ok=True)
-
- df_len = pd.read_csv(contig_to_length_tsv, sep='\t', index_col="seqid")
-
- bin_dir_stat_file_tuples = check_input_files_consistency(bin_dirs, checkm_results)
-
- logging.info(f'Consolidating {len(bin_dir_stat_file_tuples)} bins sets.')
-
- df_bins_iter = (
- parse_bin_set_stats(bin_dir, stat, min_complet, max_conta)
- for bin_dir, stat in bin_dir_stat_file_tuples
- )
-
- final_bins_set_stat = reduce(lambda b1, b2 : consolidate_two_bins_sets(b1, b2, df_len, min_overlap), df_bins_iter)
- if len(final_bins_set_stat) == 0:
- logging.warning(f"None of the bins meet minimum completeness and maximun contamination thresholds")
- sys.exit(255)
-
- logging.info('Writting final bins set ')
-
- final_bins_set_stat['genome'] = 'bins.' + final_bins_set_stat.reset_index().index.astype(str)
-
- # cleaning bin stat columns
- final_bins_set_stat = final_bins_set_stat.drop(["bin_file", "score", "contigs"], axis=1)
- final_bins_set_stat = final_bins_set_stat[~final_bins_set_stat.index.duplicated(keep='first')]
-
- logging.info(f'Writing final bin set in : {out_bin}')
- for bin_file in final_bins_set_stat.index:
-
- new_bin_id = final_bins_set_stat.loc[bin_file, "genome"]
- outfile = os.path.join(out_bin, f"{new_bin_id}.fa")
- shutil.copyfile(bin_file, outfile)
-
- logging.info(f'Writing final bin set stats: {out_bin_stats}')
- final_bins_set_stat.to_csv(out_bin_stats, sep="\t", index=False)
-
-if __name__ == "__main__":
- main()
diff --git a/bin/metawrap/dereplicate_contigs.py b/bin/metawrap/dereplicate_contigs.py
deleted file mode 100755
index 4c497e9..0000000
--- a/bin/metawrap/dereplicate_contigs.py
+++ /dev/null
@@ -1,74 +0,0 @@
-#!/usr/bin/env python3
-
-"""
-From a list of directory of sequences fasta files, get a uniq file containing all sequences once.
-
-:Example:
-python template.py -v
-"""
-
-# Metadata
-__author__ = 'Mainguy Jean - Plateforme bioinformatique Toulouse'
-__copyright__ = 'Copyright (C) 2022 INRAE'
-__license__ = 'GNU General Public License'
-__version__ = '0.1'
-__email__ = 'support.bioinfo.genotoul@inra.fr'
-__status__ = 'dev'
-
-
-from argparse import ArgumentParser, ArgumentDefaultsHelpFormatter
-import logging
-from Bio import SeqIO
-import os
-
-
-def parse_arguments():
- """Parse script arguments."""
- parser = ArgumentParser(description="...",
- formatter_class=ArgumentDefaultsHelpFormatter)
-
- parser.add_argument('--seq_dirs', nargs='+')
- parser.add_argument("-o", "--outfile", default='uniq_concat_sequences.fasta' )
- parser.add_argument("-v", "--verbose", help="increase output verbosity",
- action="store_true")
- parser.add_argument('-x', '--extension', help='Extension of input files. [Default: .fa]', default='.fa')
-
- args = parser.parse_args()
- return args
-
-
-def main():
-
- args = parse_arguments()
-
- if args.verbose:
- logging.basicConfig(format="%(levelname)s: %(message)s", level=logging.DEBUG)
- logging.info('Mode verbose ON')
-
- else:
- logging.basicConfig(format="%(levelname)s: %(message)s")
-
-
- fasta_extension = args.extension
-
- all_seq_files = []
- for seq_dir in args.seq_dirs:
- all_seq_files += [os.path.join(seq_dir, seq_file) for seq_file in os.listdir(seq_dir) if seq_file.endswith(fasta_extension)]
-
- logging.info(f"Found {len(all_seq_files)} seq files to parse.")
-
- seqs_processed = []
- with open(args.outfile, 'w') as f_out:
- for seq_file in all_seq_files:
-
- with open(seq_file, "r") as handle:
- for record in SeqIO.parse(handle, "fasta"):
- if record.id not in seqs_processed:
-
- SeqIO.write(record, f_out, "fasta")
- seqs_processed.append(record.id)
-
- logging.info(f'Have written {len(seqs_processed)} uniq sequences in {args.outfile}')
-
-if __name__ == '__main__':
- main()
diff --git a/bin/metawrap/dereplicate_contigs_in_bins.py b/bin/metawrap/dereplicate_contigs_in_bins.py
deleted file mode 100755
index d3d0465..0000000
--- a/bin/metawrap/dereplicate_contigs_in_bins.py
+++ /dev/null
@@ -1,150 +0,0 @@
-#!/usr/bin/env python3
-
-"""
-Dereplicate contigs in bins.
-
-When a contig is found in multiple bins, it is kept only in the best bin or it is remove from the bins set when --remove flag is on.
-
-:Example:
-dereplicate_contigs_in_bins.py --bins_stat binsM.stats --bin_dir binsM --out_bin_dir binsO
-"""
-
-# Metadata
-__author__ = "Mainguy Jean - Plateforme bioinformatique Toulouse"
-__copyright__ = "Copyright (C) 2022 INRAE"
-__license__ = "GNU General Public License"
-__version__ = "0.1"
-__email__ = "support.bioinfo.genotoul@inra.fr"
-__status__ = "dev"
-
-
-from argparse import ArgumentParser, ArgumentDefaultsHelpFormatter
-import logging
-from Bio import SeqIO
-import os
-import pandas as pd
-import sys
-from collections import defaultdict
-
-
-def parse_arguments():
- """Parse script arguments."""
- parser = ArgumentParser(
- description="...", formatter_class=ArgumentDefaultsHelpFormatter
- )
-
- parser.add_argument(
- "-r",
- "--bins_stat",
- required=True,
- help="bin set stat file with completeness, contamination and N50 metrics.",
- )
-
- parser.add_argument("-b", "--bin_dir", required=True, help="Bin set directory.")
-
- parser.add_argument(
- "-o",
- "--out_bin_dir",
- required=True,
- )
- parser.add_argument(
- "-x",
- "--extension",
- help="Extension of input files. [Default: fa]",
- default="fa",
- )
-
- parser.add_argument(
- "--remove",
- help="Remove contigs that end up in more than one bin",
- action="store_true",
- )
-
- parser.add_argument(
- "-v", "--verbose", help="increase output verbosity", action="store_true"
- )
-
- args = parser.parse_args()
- return args
-
-
-def get_contig2bins(fasta_bins):
-
- contig2bins = defaultdict(list)
-
- for seq_file in fasta_bins:
- bin_id = ".".join(os.path.basename(seq_file).split(".")[:-1])
- for record in SeqIO.parse(seq_file, "fasta"):
- contig2bins[record.id].append(bin_id)
-
- return contig2bins
-
-
-def main():
-
- args = parse_arguments()
-
- if args.verbose:
- logging.basicConfig(format="%(levelname)s: %(message)s", level=logging.DEBUG)
- logging.info("Mode verbose ON")
-
- else:
- logging.basicConfig(format="%(levelname)s: %(message)s")
-
- bins_stats = args.bins_stat
- bin_dir = args.bin_dir
- out_bin_dir = args.out_bin_dir
- fasta_extension = args.extension
- remove_flag = args.remove
-
- os.makedirs(out_bin_dir, exist_ok=True)
-
- logging.info("Loading bin completion and contamination scores...")
- df = pd.read_csv(bins_stats, sep="\t", index_col="genome")
- df["score"] = df["completeness"] - 5 * df["contamination"] + 1e-10 * df["N50"]
-
-
-
- logging.info("Loading in contigs in each bin...")
-
- bin_files = [
- os.path.join(bin_dir, seq_file)
- for seq_file in os.listdir(bin_dir)
- if seq_file.endswith(fasta_extension)
- ]
-
- contig2bins = get_contig2bins(bin_files)
-
-
- logging.info("Get dereplicated contig list per bin...")
-
- bin2uniqcontigs = defaultdict(list)
-
- for contig, bins in contig2bins.items():
- if len(bins) > 1 and remove_flag:
- logging.info(
- f"{contig} is removed because it is found in more than one bin. (remove flag is on)"
- )
- continue
-
- best_bin = df.loc[bins, "score"].idxmax()
-
- bin2uniqcontigs[best_bin].append(contig)
-
- # go over the bin files again and make a new dereplicated version of each bin file
- logging.info(
- f"Making a new dereplicated version of each bin file and writting them in {out_bin_dir}"
- )
- for bin_id, contigs in bin2uniqcontigs.items():
-
- bin_file = os.path.join(bin_dir, f"{bin_id}.{fasta_extension}")
- out_bin_file = os.path.join(out_bin_dir, f"{bin_id}.{fasta_extension}")
-
- with open(bin_file) as in_fh, open(out_bin_file, "w") as out_fh:
- for record in SeqIO.parse(in_fh, "fasta"):
- if record.id in contigs:
- SeqIO.write(record, out_fh, "fasta")
-
-
-if __name__ == "__main__":
- main()
diff --git a/bin/metawrap/get_contig_length.py b/bin/metawrap/get_contig_length.py
deleted file mode 100755
index 22ad5fa..0000000
--- a/bin/metawrap/get_contig_length.py
+++ /dev/null
@@ -1,73 +0,0 @@
-#!/usr/bin/env python3
-
-"""
-Get lengths of contigs
-
-:Example:
-python template.py -v
-"""
-
-# Metadata
-__author__ = 'Mainguy Jean - Plateforme bioinformatique Toulouse'
-__copyright__ = 'Copyright (C) 2022 INRAE'
-__license__ = 'GNU General Public License'
-__version__ = '0.1'
-__email__ = 'support.bioinfo.genotoul@inra.fr'
-__status__ = 'dev'
-
-
-from argparse import ArgumentParser, ArgumentDefaultsHelpFormatter
-import logging
-from Bio import SeqIO
-import os
-
-
-def parse_arguments():
- """Parse script arguments."""
- parser = ArgumentParser(description="...",
- formatter_class=ArgumentDefaultsHelpFormatter)
-
- parser.add_argument('--seq_dirs', nargs='+')
- parser.add_argument("-o", "--outfile", default='contig_to_length.tsv' )
- parser.add_argument("-v", "--verbose", help="increase output verbosity",
- action="store_true")
- parser.add_argument('-x', '--extension', help='Extension of input files. [Default: .fa]', default='.fa')
-
- args = parser.parse_args()
- return args
-
-
-def main():
-
- args = parse_arguments()
-
- if args.verbose:
- logging.basicConfig(format="%(levelname)s: %(message)s", level=logging.DEBUG)
- logging.info('Mode verbose ON')
-
- else:
- logging.basicConfig(format="%(levelname)s: %(message)s")
-
-
- fasta_extension = args.extension
-
- seqid2length = {}
- all_seq_files = []
- for seq_dir in args.seq_dirs:
- all_seq_files += [os.path.join(seq_dir, seq_file) for seq_file in os.listdir(seq_dir) if seq_file.endswith(fasta_extension)]
-
- logging.info(f"Found {len(all_seq_files)} seq files to parse.")
-
- for seq_file in all_seq_files:
- with open(seq_file, "r") as handle:
- for record in SeqIO.parse(handle, "fasta"):
- if record.id not in seqid2length:
- seqid2length[record.id] = len(record.seq)
-
- seqid2length_iter = (f"{seqid}\t{length}" for seqid, length in seqid2length.items())
- with open(args.outfile, 'w') as fl:
- fl.write(f"seqid\tlength\n")
- fl.write('\n'.join(seqid2length_iter))
-
-if __name__ == '__main__':
- main()
diff --git a/bin/metawrap/plot_binning_results.py b/bin/metawrap/plot_binning_results.py
deleted file mode 100755
index ef1a133..0000000
--- a/bin/metawrap/plot_binning_results.py
+++ /dev/null
@@ -1,246 +0,0 @@
-#!/usr/bin/env python3
-
-"""
-This script is coming from metaWRAP bin_refinement pipeline.
-
-We thank the author of the orginal metawrap: German Uritskiy.
-Check metaWRAP github: https://github.com/bxlab/metaWRAP
-"""
-
-
-# USAGE:
-# ./script file1.stats file2.stats file3.stats
-
-import sys
-import matplotlib.pyplot as plt
-plt.switch_backend('agg')
-
-max_contamination=int(sys.argv[2])
-min_completion=int(sys.argv[1])
-
-####################################################################################################################################
-############################################ MAKE THE COMPLETION PLOT ############################################
-####################################################################################################################################
-print("Loading completion info....")
-data={}
-max_x=0
-# loop over all bin .stats files
-for file_name in sys.argv[3:]:
- print(file_name)
- bin_set=".".join(file_name.split("/")[-1].split(".")[:-1])
- data[bin_set]=[]
- for line in open(file_name):
- # skip header
- if "compl" in line: continue
-
- # skip bins that are too contaminated or very incomplete
- if float(line.split("\t")[2])>max_contamination: continue
- if float(line.split("\t")[1])<min_completion: continue
-
- # save the completion value of each bin into a list
- data[bin_set].append(float(line.split("\t")[1]))
- if len(data[bin_set])>max_x: max_x=len(data[bin_set])
-
-# sort the completion data sets
-for bin_set in data:
- data[bin_set].sort(reverse=True)
-
-print("Plotting completion data...")
-# MAKING THE PLOT PRETTY!!!!
-# set some color schemes
-tableau20 = [(214, 39, 40), (31, 119, 180), (255, 127, 14),
- (44, 160, 44), (255, 152, 150),
- (148, 103, 189), (197, 176, 213), (140, 86, 75), (196, 156, 148),
- (227, 119, 194), (247, 182, 210), (127, 127, 127), (199, 199, 199),
- (188, 189, 34), (219, 219, 141), (23, 190, 207), (158, 218, 229)]
-
-for i in range(len(tableau20)):
- r, g, b = tableau20[i]
- tableau20[i] = (r / 255., g / 255., b / 255.)
-plot_colors={}
-for i, label in enumerate(sys.argv[1:]):
- bin_set=".".join(label.split("/")[-1].split(".")[:-1])
- plot_colors[bin_set]=tableau20[i]
-
-
-
-# set figure size
-plt.figure(figsize=(16, 8))
-plt.style.use('ggplot')
-
-# Remove the plot frame lines. They are unnecessary chartjunk.
-ax = plt.subplot(121)
-ax.spines["top"].set_visible(False)
-ax.spines["bottom"].set_linewidth(0.5)
-ax.spines['bottom'].set_color('black')
-ax.spines["right"].set_visible(False)
-ax.spines["left"].set_visible(False)
-#ax.set_facecolor('white')
-ax.set_facecolor("white")
-
-# Ensure that the axis ticks only show up on the bottom and left of the plot.
-ax.get_xaxis().tick_bottom()
-ax.get_yaxis().tick_left()
-
-# Limit the range of the plot to only where the data is.
-plt.ylim(min_completion, 105)
-max_x=0
-for k in data:
- if len(data[k])>max_x: max_x=len(data[k])
-plt.xlim(0, max_x)
-
-# Make sure your axis ticks are large enough to be easily read.
-plt.yticks(list(range(min_completion, 105, 10)), [str(x) + "%" for x in range(min_completion, 105, 10)], fontsize=14)
-plt.xticks(fontsize=14)
-
-# Provide tick lines across the plot to help your viewers trace along
-for y in range(min_completion, 105, 10):
- plt.axhline(y=y, linestyle="--", lw=0.5, color="black", alpha=0.3)
-for x in range(0, 1000, 20):
- plt.axvline(x=x, linestyle="--", lw=0.5, color="black", alpha=0.3)
-
-# Remove the tick marks; they are unnecessary with the tick lines we just plotted.
-plt.tick_params(axis="both", which="both", bottom=False, top=False, labelbottom=True, left=False, right=False, labelleft=True)
-
-
-# PLOTTING THE DATA
-
-# prepare labeles
-labels = []
-for k in data: labels.append(k)
-
-# plot the data and labels
-N = len(labels)
-y_increment = (100-min_completion)/N/2
-y_pos = 100-y_increment
-
-for rank, bin_set in enumerate(labels):
- # chose a color!
- c=plot_colors[bin_set]
-
- # plot the data
- plt.plot(data[bin_set], lw=2.5, color=c)
-
- # add bin set label to plot
- for x_pos,y in enumerate(data[bin_set]):
- if y<y_pos:
- break
- plt.text(x_pos, y_pos, bin_set, fontsize=18, color=c)
- y_pos-=y_increment
-
-# add plot and axis titles and adjust edges
-plt.title("Bin completion ranking", fontsize=26)
-plt.xlabel("Descending completion rank", fontsize=16)
-plt.ylabel("Estimated bin completion", fontsize=16)
-
-
-
-
-####################################################################################################################################
-############################################ MAKE THE CONTAMINATION PLOT ############################################
-####################################################################################################################################
-print("Loading contamination info...")
-
-data={}
-# loop over all bin .stats files
-for file_name in sys.argv[3:]:
- bin_set=".".join(file_name.split("/")[-1].split(".")[:-1])
- data[bin_set]=[]
- for line in open(file_name):
- # skip header
- if "compl" in line: continue
-
- # skip bins that are too incomplete or way too contaminated
- if float(line.split("\t")[1])<min_completion: continue
- if float(line.split("\t")[2])>max_contamination: continue
-
- # save the contamination value of each bin into a list
- data[bin_set].append(float(line.split("\t")[2]))
-
-# sort the contamination data sets
-for bin_set in data:
- data[bin_set].sort(reverse=False)
-
-print("Plotting the contamination data...")
-# MAKING THE PLOT PRETTY!!!!
-# Remove the plot frame lines. They are unnecessary chartjunk.
-ax = plt.subplot(122)
-ax.spines["top"].set_visible(False)
-ax.spines["bottom"].set_linewidth(0.5)
-ax.spines['bottom'].set_color('black')
-ax.spines["right"].set_visible(False)
-ax.spines["left"].set_visible(False)
-#ax.set_facecolor('white')
-ax.set_facecolor("white")
-
-# Ensure that the axis ticks only show up on the bottom and left of the plot.
-ax.get_xaxis().tick_bottom()
-ax.get_yaxis().tick_left()
-
-# Limit the range of the plot to only where the data is.
-#plt.gca().invert_yaxis()
-plt.ylim(0, max_contamination+1)
-#ax.set_yscale('log')
-max_x=0
-for k in data:
- if len(data[k])>max_x: max_x=len(data[k])
-plt.xlim(0, max_x)
-
-# Make sure your axis ticks are large enough to be easily read.
-plt.yticks(list(range(-0, max_contamination+1, 1)), [str(x) + "%" for x in range(-0, max_contamination+1, 1)], fontsize=14)
-plt.xticks(fontsize=14)
-
-# Provide tick lines across the plot to help your viewers trace along
-for y in range(0, max_contamination+1, 1):
- plt.axhline(y=y, linestyle="--", lw=0.5, color="black", alpha=0.3)
-for x in range(0, 1000, 20):
- plt.axvline(x=x, linestyle="--", lw=0.5, color="black", alpha=0.3)
-
-
-# Remove the tick marks; they are unnecessary with the tick lines we just plotted.
-plt.tick_params(axis="both", which="both", bottom=False, top=False, labelbottom=True, left=False, right=False, labelleft=True)
-
-
-# PLOTTING THE DATA
-# prepare labeles
-labels = []
-for k in data: labels.append(k)
-
-# plot the data and labels
-N = len(labels)
-y_increment = max_contamination/N/2
-y_pos = y_increment
-
-for rank, bin_set in enumerate(labels):
- # chose a color!
- c=plot_colors[bin_set]
-
- # plot the data
- plt.plot(data[bin_set], lw=2.5, color=c)
-
- # add bin set label to plot
- for x_pos,y in enumerate(data[bin_set]):
- if y>y_pos:
- break
- plt.text(x_pos, y_pos, bin_set, fontsize=18, color=c)
- y_pos+=y_increment
-
-
-# add plot and axis titles and adjust the edges
-plt.title("Bin contamination ranking", fontsize=26)
-plt.xlabel("Acending contamination rank", fontsize=16)
-plt.ylabel("Estimated bin contamination (log scale)", fontsize=16)
-plt.gcf().subplots_adjust(right=0.9)
-
-# save figure
-print("Saving figures binning_results.eps and binning_results.png ...")
-plt.tight_layout(w_pad=10)
-plt.subplots_adjust(top=0.92, right=0.90, left=0.08)
-plt.savefig("binning_results.png",format='png', dpi=300)
-#plt.show()
-
-
-
-
-
-
diff --git a/bin/metawrap/print_comment.py b/bin/metawrap/print_comment.py
deleted file mode 100755
index be1ef7d..0000000
--- a/bin/metawrap/print_comment.py
+++ /dev/null
@@ -1,36 +0,0 @@
-#!/usr/bin/env python3
-
-"""
-This script is coming from metaWRAP bin_refinement pipeline.
-
-We thank the author of the orginal metawrap: German Uritskiy.
-Check metaWRAP github: https://github.com/bxlab/metaWRAP
-"""
-
-# This script prints any comment in a structured and prety way.
-import sys
-comm=sys.argv[1]
-delim=sys.argv[2]
-
-print('\n'+delim*120)
-
-max_len=90
-
-cut=comm.split(" ")
-line=""
-for word in cut:
- if (len(line) + 1 + len(word))>max_len:
- edge1=int((120-len(line))/2 - 5)
- edge2=120-edge1-len(line) - 10
- print(delim*5 + " "*edge1 + line + " "*edge2 + delim*5)
- line=word
- else:
- line = line+" "+word
-edge1=int((120-len(line))/2 - 5)
-edge2=120-edge1-len(line) - 10
-print(delim*5 + " "*edge1 + line + " "*edge2 + delim*5)
-
-print(delim*120+'\n')
-
-
-
diff --git a/bin/metawrap/setup_indermediate_checkm2_files.py b/bin/metawrap/setup_indermediate_checkm2_files.py
deleted file mode 100644
index 094d7fd..0000000
--- a/bin/metawrap/setup_indermediate_checkm2_files.py
+++ /dev/null
@@ -1,171 +0,0 @@
-#!/usr/bin/env python3
-
-"""
-Setup intermediate files of checkm2 of bin sets
-to skip prodigal and diamond steps in checkm2
-
-:Example:
-python template.py -v
-"""
-
-# Metadata
-__author__ = 'Mainguy Jean - Plateforme bioinformatique Toulouse'
-__copyright__ = 'Copyright (C) 2022 INRAE'
-__license__ = 'GNU General Public License'
-__version__ = '0.1'
-__email__ = 'support.bioinfo.genotoul@inra.fr'
-__status__ = 'dev'
-
-
-from argparse import ArgumentParser, ArgumentDefaultsHelpFormatter
-import logging
-from Bio import SeqIO
-import os
-from collections import defaultdict
-import os
-import errno
-import sys
-import pandas as pd
-
-def parse_arguments():
- """Parse script arguments."""
- parser = ArgumentParser(description="...",
- formatter_class=ArgumentDefaultsHelpFormatter)
-
- parser.add_argument('-b', '--bin_dir', required=True, help="Bin directory.",)
- parser.add_argument('-f', '--faa_raw', required=True, help="Protein sequences predicted with prodigal on all contigs.",)
- parser.add_argument('-d', '--diamond_raw', required=True, help="Results from diamond launched on all contigs.",)
- parser.add_argument("-o", "--outdir", required=True)
- parser.add_argument("-v", "--verbose", help="increase output verbosity",
- action="store_true")
- parser.add_argument('-x', '--extension', help='Extension of input files. [Default: .fa]', default='.fa')
-
- args = parser.parse_args()
- return args
-
-def get_contig2bin(fasta_bins):
-
- contig2bin = {}
-
- for seq_file in fasta_bins:
- bin_id = '.'.join(os.path.basename(seq_file).split('.')[:-1])
- contig2bin.update({record.id:bin_id for record in SeqIO.parse(seq_file, "fasta")})
-
- return contig2bin
-
-def make_sure_path_exists(path):
- """
- Create directory if it does not exist.
-
- Taken from Checkm2. https://github.com/chklovski/CheckM2/blob/e563159f005344d798defb60cea4189e0be8ec92/checkm2/fileManager.py#L218
- """
-
- if not path:
- return
-
- try:
- os.makedirs(path)
- except OSError as exception:
- if exception.errno != errno.EEXIST:
- logging.error('Specified path does not exist: ' + path + '\n')
- sys.exit(1)
-
-def from_faa_to_bin(faa_id, contig2bin):
- contig = "_".join(faa_id.split('_')[:-1])
-
- try:
- bin_id = contig2bin[contig]
- except KeyError:
- bin_id = None
-
- return bin_id
-
-def get_bin2faa_seq(faa_file, contig2bin):
-
- bin2faa_seq = defaultdict(list)
- contigs_not_in_bin = set()
- # load all faa sequences into memory and make a bin2sequencerecord
- for record in SeqIO.parse(faa_file, "fasta"):
-
- contig = '_'.join(record.id.split('_')[:-1])
-
- if contig not in contig2bin:
- contigs_not_in_bin.add(contig)
- continue
-
- bin_id = contig2bin[contig]
- bin2faa_seq[bin_id].append(record)
-
- logging.info(f"{len(contigs_not_in_bin)} contigs have been ignored as they do not belong to any bins.")
- return bin2faa_seq
-
-def main():
-
- args = parse_arguments()
-
- if args.verbose:
- logging.basicConfig(format="%(levelname)s: %(message)s", level=logging.DEBUG)
- logging.info('Mode verbose ON')
-
- else:
- logging.basicConfig(format="%(levelname)s: %(message)s")
-
- bin_dir = args.bin_dir
- fasta_extension = args.extension
-
- diamond_outname = "DIAMOND_RESULTS.tsv"
-
- diamond_raw = args.diamond_raw
-
- prodigal_outdir_name = "protein_files"
- diamond_outdir_name = "diamond_output"
-
- diamond_header_separator = 'Ω'
-
- prodigal_out_path = os.path.join(args.outdir, prodigal_outdir_name)
- diamond_out_path = os.path.join(args.outdir, diamond_outdir_name)
-
- make_sure_path_exists(prodigal_out_path)
- make_sure_path_exists(diamond_out_path)
-
-
- diamond_output = os.path.join(diamond_out_path, diamond_outname)
-
-
- logging.info("Get bin composition by parsing bin fasta files.")
-
- bin_files = [os.path.join(bin_dir, seq_file) for seq_file in os.listdir(bin_dir) if seq_file.endswith(fasta_extension)]
- contig2bin = get_contig2bin(bin_files)
-
- logging.info("Setting up faa output.")
-
- bin2faa_seq = get_bin2faa_seq(args.faa_raw, contig2bin)
-
- logging.info(f"Writting faa into {len(bin2faa_seq)} bin files in {prodigal_out_path}.")
-
- for bin_id, faa_seq in bin2faa_seq.items():
-
- bin_out_file = os.path.join(prodigal_out_path, f'{bin_id}.faa')
- SeqIO.write(faa_seq, bin_out_file, "fasta")
-
- logging.info("Setting up DIAMOND output.")
-
- headers = ['query_id', "subject_id"] + [f"_{i}" for i in range(10)]
- df = pd.read_csv(diamond_raw, sep='\t', names= headers)
-
- # Get bin id for each query id
- df['bin'] = df['query_id'].apply(lambda x: from_faa_to_bin(x,contig2bin))
-
- # remove line that are from query that does not belong to current bin set
- df = df.loc[ ~ df['bin'].isna()]
-
- # concat binid and query id with checkm2 separator
- df['query_id'] = df['bin'] + diamond_header_separator + df['query_id']
- df = df.drop('bin', axis=1)
-
- df.to_csv(diamond_output, sep="\t", index=False, header=False)
-
- return
-
-if __name__ == '__main__':
- main()
diff --git a/conf/base.config b/conf/base.config
index 721713b..8bac4a7 100644
--- a/conf/base.config
+++ b/conf/base.config
@@ -100,7 +100,7 @@ process {
memory = { 30.GB * task.attempt }
cpus = 20
}
- withName: METAWRAP_REFINMENT {
+ withName: BINETTE {
memory = { 20.GB * task.attempt }
cpus = 20
}
diff --git a/conf/test.config b/conf/test.config
index 8b437cd..1a57d0d 100644
--- a/conf/test.config
+++ b/conf/test.config
@@ -103,7 +103,7 @@ process {
memory = { 1.GB * task.attempt }
executor = "local"
}
- withName: METAWRAP_REFINMENT {
+ withName: BINETTE {
memory = { 12.GB * task.attempt }
cpus = 8
}
diff --git a/docs/output.md b/docs/output.md
index ea47fa6..f5622a6 100644
--- a/docs/output.md
+++ b/docs/output.md
@@ -240,7 +240,7 @@ The `results/` directory contains a sub-directory for each step launched:
| `SAMPLE_NAME/concoct`| This directory contains bins fasta files from the individual binning tool CONCOCT (see informations [here](https://concoct.readthedocs.io/en/latest/)). |
| `SAMPLE_NAME/maxbin2`| This directory contains bins fasta files from the individual binning tool MaxBin2(see informations [here](https://academic.oup.com/bioinformatics/article/32/4/605/1744462)). |
| `SAMPLE_NAME/metabat2`| This directory contains bins fasta files from the individual binning tool MetaBAT2 (see informations [here](https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6662567/)). |
-| `SAMPLE_NAME/bin_refinement`| This directory contains bins fasta files from the aggregate binning module bin_refinement from metaWRAP tool, which takes into account the bins sets from the 3 individual binning tools to try to improve the quality of the bins (see informations [here](https://microbiomejournal.biomedcentral.com/articles/10.1186/s40168-018-0541-1)). |
+| `SAMPLE_NAME/bin_refinement`| This directory contains bins fasta files and statistics from the aggregate binning tool BINETTE, which takes into account the bins sets from the 3 individual binning tools to try to improve the quality of the bins (see informations [here](https://forgemia.inra.fr/jean.mainguy/binette)). |
| `SAMPLE_NAME/bin_refinement/unbinned_contigs.fasta`| Contigs from the sample assembly that are not retrieved in any bin. The proportion of non-binned contigs to the global size of the sample assembly can be seen in the MultiQC report, in the section Bins Size (bp) quality. |
#### **08_binning/08_2_dereplicated_bins**
diff --git a/env/Singularity_recipe_binning b/env/Singularity_recipe_binning
index 00f6e2f..4221b25 100644
--- a/env/Singularity_recipe_binning
+++ b/env/Singularity_recipe_binning
@@ -6,16 +6,22 @@ IncludeCmd: yes
env/binning.yml /
%post
-apt-get update && apt-get install -y procps && apt-get clean -y
-/opt/conda/bin/mamba env create -f /binning.yml && /opt/conda/bin/conda clean -a
+# build-essential g++ are needed to pip install checkm2
+apt-get update && apt-get install -y procps build-essential g++ && apt-get clean -y
+/opt/conda/bin/mamba env create -f /binning.yml && /opt/conda/bin/conda clean -a
-git clone --recursive https://github.com/JeanMainguy/CheckM2.git
+export PATH=/opt/conda/envs/binning/bin:$PATH
+git clone https://forgemia.inra.fr/jean.mainguy/binette.git
-export PATH=/opt/conda/envs/binning/bin:/CheckM2/bin/:$PATH
-checkm2 database --download --path '.'
+git clone --recursive https://github.com/chklovski/checkm2.git
+
+pip install checkm2/
+
+checkm2 database --download --path '.' || echo checkm2 database failed but it is probably ok
%environment
-export PATH=/opt/conda/envs/binning/bin:/CheckM2/bin/:$PATH
+export PATH=/opt/conda/envs/binning/bin/:/checkm2/bin/:/binette/binette/:$PATH
%runscript
"$@"
+
diff --git a/env/binning.yml b/env/binning.yml
index ead356b..7577e2b 100644
--- a/env/binning.yml
+++ b/env/binning.yml
@@ -1,16 +1,9 @@
name: binning
channels:
- - conda-forge
- bioconda
- defaults
+ - conda-forge
dependencies:
- - biopython
- - matplotlib
- - metabat2=2.15
- - maxbin2=2.2.7
- - concoct=1.1.0
- - gtdbtk=2.1
- - drep=3.0.0
- python>=3.6
- scikit-learn=0.23.2
- h5py=2.10.0
@@ -21,8 +14,18 @@ dependencies:
- pandas
- scipy
- prodigal>=2.6.3
- - pprodigal
- setuptools
- requests
- packaging
- tqdm
+ - networkx
+ - pyfastx
+ - pyrodigal
+ - biopython
+ - matplotlib
+ - metabat2=2.15
+ - maxbin2=2.2.7
+ - concoct=1.1.0
+ - gtdbtk=2.1
+ - drep=3.0.0
+ - pprodigal
diff --git a/modules/binning.nf b/modules/binning.nf
index 431fb01..9cc6879 100644
--- a/modules/binning.nf
+++ b/modules/binning.nf
@@ -112,37 +112,40 @@ process CONCOCT {
}
-process METAWRAP_REFINMENT {
+process BINETTE {
errorStrategy { task.exitStatus == 255 ? 'ignore' : 'finish' }
tag "${meta.id}"
publishDir "${params.outdir}/08_binning/08_1_binning_per_sample/${meta.id}", mode: 'copy'
label 'BINNING'
input:
- tuple val(meta), val(bins1), val(bins2), val(bins3)
+ tuple val(meta), val(bins), path(contigs)
val min_completeness
val max_contamination
output:
- path "bin_refinement/figures/*"
- tuple val(meta), path("bin_refinement/metawrap_*_bins/*"), emit: bins
- tuple val(meta), path('bin_refinement/metawrap_*_bins.stats'), emit: checkm_stats
- path "v_metawrap.txt", emit: v_metawrap
+ tuple val(meta), path("bin_refinement/final_bins/*"), emit: bins
+ tuple val(meta), path('bins_stats.tsv'), emit: checkm_stats
+ path "v_binette.txt", emit: v_binette
script:
- bins_flag = (bins3 != null) ? "-A $bins1 -B $bins2 -C $bins3" : "-A $bins1 -B $bins2 "
"""
+ binette.py -v -m 0 -t ${task.cpus} -d ${bins.join(' ')} -c ${contigs} -o bin_refinement/
- echo "metawrap 1.3_modified" > v_metawrap.txt
-
- bin_refinement.sh -t ${task.cpus} -c $min_completeness -x $max_contamination -o bin_refinement/ $bins_flag
- cd bin_refinement/metawrap_${min_completeness}_${max_contamination}_bins/
- for filename in *fa;
- do
- mv \$filename "${meta.id}_\$filename";
- sed -i 's/^bin/${meta.id}_bin/' ../metawrap_${min_completeness}_${max_contamination}_bins.stats
- done
+ sed -i 's/size/Size/' bin_refinement/final_bins_quality_reports.tsv
+ sed -i 's/^/${meta.id}_bin_/' bin_refinement/final_bins_quality_reports.tsv
+ sed -i 's/^${meta.id}_bin_bin_id/genome/' bin_refinement/final_bins_quality_reports.tsv
+ cut -f 2,3 --complement bin_refinement/final_bins_quality_reports.tsv > bins_stats.tsv
+
+ cd bin_refinement/final_bins
+
+ for filename in *fa;
+ do
+ mv \$filename "${meta.id}_\$filename"
+ done
+ cd ../..
+ touch v_binette.txt
"""
}
diff --git a/modules/checkm2.nf b/modules/checkm2.nf
index 08b4376..94fc69c 100644
--- a/modules/checkm2.nf
+++ b/modules/checkm2.nf
@@ -10,7 +10,7 @@ process CHECKM2 {
script:
"""
- checkm2 predict -x fa --threads ${task.cpus} --input $bins --output-directory checkm2/
+ checkm2 predict -x fa --threads ${task.cpus} --input ${bins.join(' ')} --output-directory checkm2/
echo "\$(checkm2 --version)_modified" > v_checkm2.txt
"""
diff --git a/modules/get_db_versions.nf b/modules/get_db_versions.nf
index d41ecbe..9805a29 100644
--- a/modules/get_db_versions.nf
+++ b/modules/get_db_versions.nf
@@ -48,7 +48,7 @@ process GET_DB_VERSIONS {
echo "GTDBTK ${gtdbtk}" > gtdbtk_db.txt
fi
- checkm2=\$(checkm2 database --current 2>&1 | awk '{ print \$5 }')
+ checkm2=\$(checkm2 database --current 2>&1 | tail -n1 |awk '{ print \$5 }')
echo "Diamond_checkm2 \$checkm2" > checkm2_db.txt
if [[ `ls | grep db.txt` ]]
diff --git a/modules/sum_up_bins_informations.nf b/modules/sum_up_bins_informations.nf
index 5cfc103..e4df705 100644
--- a/modules/sum_up_bins_informations.nf
+++ b/modules/sum_up_bins_informations.nf
@@ -52,7 +52,7 @@ process ADD_QUAST_INFO_TO_BINS {
val=i;}}}
if(val != -1) print \$1 "\t" \$val} ' report.tsv > report_${meta.id}.tsv
- add_info_to_metawrap_stat.py -s $bins_stat \
+ add_info_to_bin_stat.py -s $bins_stat \
-q report_${meta.id}.tsv \
-o "${meta.id}_bins_stat_and_quality.tsv"
"""
diff --git a/subworkflows/08_binning.nf b/subworkflows/08_binning.nf
index 69a7962..df6ca95 100644
--- a/subworkflows/08_binning.nf
+++ b/subworkflows/08_binning.nf
@@ -1,4 +1,4 @@
-include { GENERATE_DEPTH_FILES; METABAT2; MAXBIN2; CONCOCT; METAWRAP_REFINMENT; UNBINNED_CONTIGS } from '../modules/binning'
+include { GENERATE_DEPTH_FILES; METABAT2; MAXBIN2; CONCOCT; BINETTE; UNBINNED_CONTIGS } from '../modules/binning'
include { CHECKM2 } from '../modules/checkm2'
include { DREP } from '../modules/drep'
include { BWA_MEM;BWA_MEM as BWA_MEM_CROSS_ALIGNMENT; MINIMAP2; MINIMAP2 as MINIMAP2_CROSS_ALIGNMENT } from '../modules/read_alignment'
@@ -25,7 +25,7 @@ workflow STEP_08_BINNING {
ch_metabat2_v = Channel.empty()
ch_maxbin_v = Channel.empty()
ch_concoct_v = Channel.empty()
- ch_metawrap_v = Channel.empty()
+ ch_binette_v = Channel.empty()
ch_checkm2_v = Channel.empty()
ch_dRep_v = Channel.empty()
ch_gtdbtk_v = Channel.empty()
@@ -140,64 +140,37 @@ workflow STEP_08_BINNING {
//////////////////
//// BIN REFINEMENT
//////////////////
- ch_bins_set = ch_metabat_bins.join(ch_concoct_bins, remainder:true)
- .join(ch_maxbin_bins, remainder:true)
- .branch{
- multiple: (it[1] != null && it[2] != null) ||
- (it[1] != null && it[3] != null) ||
- (it[2] != null && it[3] != null)
- single: true
- }
-
- //bins set with bins form at least 2 binners
- ch_bins_multiple = ch_bins_set.multiple.map{ meta, bins1, bins2, bins3 ->
- if ( bins1 == null ) {
- return [meta, bins2, bins3, bins1]
- } else if ( bins2 == null ) {
- return [meta, bins1, bins3, bins2]
- } else {
- return [meta, bins1, bins2, bins3]
- }
- }
-
- //bins set with bins form only 1 binner
- ch_bins_single = ch_bins_set.single.map{ meta, bins1, bins2, bins3 ->
- if ( bins1 != null ) {
- return [meta, bins1]
- } else if ( bins2 != null ) {
- return [meta, bins2]
- } else {
- return [meta, bins3]
- }
- }
-
- CHECKM2(ch_bins_single)
+ ch_bins_set = ch_metabat_bins.mix(ch_concoct_bins, ch_maxbin_bins)
+ .groupTuple(by: [0])
+ .branch{ meta, bins ->
+ multiple: bins.size() > 1
+ single: bins.size() == 1
+ }
+
+ CHECKM2(ch_bins_set.single)
ch_checkm2_v = CHECKM2.out.v_checkm2
ch_all_bins_stats = Channel.empty()
- ch_metawrap_stats_and_quast = Channel.empty()
- ch_metawrap_bins = Channel.empty()
- ch_metawrap_bins = Channel.empty()
- ch_metawrap_stats_and_quast = Channel.empty()
+ ch_binette_stats_and_quast = Channel.empty()
+ ch_binette_bins = Channel.empty()
+ ch_binette_stats = Channel.empty()
ch_bins_assembly = Channel.empty()
- METAWRAP_REFINMENT(ch_bins_multiple, params.min_completeness, params.max_contamination)
- ch_metawrap_bins = METAWRAP_REFINMENT.out.bins
- .map { meta, file -> file}
- .collect()
- ch_metawrap_stats = METAWRAP_REFINMENT.out.checkm_stats
- .map { meta, file -> file}
- .collect()
+ ch_bins_assembly = ch_bins_set.multiple.join(ch_assembly)
+
+ BINETTE(ch_bins_assembly, params.min_completeness, params.max_contamination)
+ ch_binette_bins = BINETTE.out.bins
+ ch_binette_stats = BINETTE.out.checkm_stats
- ch_bins_assembly = METAWRAP_REFINMENT.out.bins.join(ch_assembly)
+ ch_bins_assembly = ch_binette_bins.join(ch_assembly)
UNBINNED_CONTIGS(ch_bins_assembly)
- ch_metawrap_stats_and_quast = METAWRAP_REFINMENT.out.checkm_stats.combine(quast)
- ch_metawrap_v = METAWRAP_REFINMENT.out.v_metawrap
+ ch_binette_stats_and_quast = ch_binette_stats.combine(quast)
+ ch_binette_v = BINETTE.out.v_binette
- ADD_QUAST_INFO_TO_BINS(ch_metawrap_stats_and_quast)
+ ADD_QUAST_INFO_TO_BINS(ch_binette_stats_and_quast)
ch_all_bins_stats = ADD_QUAST_INFO_TO_BINS.out.bins_stats.collect()
@@ -211,8 +184,13 @@ workflow STEP_08_BINNING {
////////////////////////
ch_bins_drep = Channel.empty()
+ ch_binette_bins = ch_binette_bins.map { meta, file -> file}
+ .collect()
+
+ ch_binette_stats = ch_binette_stats.map { meta, file -> file}
+ .collect()
- DREP(ch_metawrap_stats, ch_metawrap_bins, params.drep_threshold)
+ DREP(ch_binette_stats, ch_binette_bins, params.drep_threshold)
ch_bins_drep = DREP.out.drep_bins_folder
ch_dRep_v = DREP.out.v_dRep
@@ -260,10 +238,10 @@ workflow STEP_08_BINNING {
ch_metabat2_v.first(),
ch_maxbin_v.first(),
ch_concoct_v.first(),
- ch_metawrap_v,
+ ch_binette_v,
ch_checkm2_v.first(),
ch_dRep_v,
- ch_gtdbtk_v,
+ ch_gtdbtk_v
)
emit:
--
GitLab
From 2c447579a5e69927f02d848748e70aea0c17dff0 Mon Sep 17 00:00:00 2001
From: Jean Mainguy <jean.mainguy@inra.fr>
Date: Wed, 18 Jan 2023 15:37:50 +0100
Subject: [PATCH 179/194] add mapping file to map original and new contig name
---
bin/rename_contigs.py | 4 +++-
docs/output.md | 3 +--
modules/assembly.nf | 3 ++-
subworkflows/02_assembly.nf | 4 +++-
4 files changed, 9 insertions(+), 5 deletions(-)
diff --git a/bin/rename_contigs.py b/bin/rename_contigs.py
index beb627b..c6556e1 100755
--- a/bin/rename_contigs.py
+++ b/bin/rename_contigs.py
@@ -37,6 +37,8 @@ def parse_arguments():
parser.add_argument('-i', '--fna_file', help='Original fasta file of contigs.', required=True)
parser.add_argument('-o', '--out_fna', help='Output fasta file with renamed contigs.', required=True)
+
+ parser.add_argument('-t', '--contig_names_table', help='Tabular table with 2 fields : orignal and new name.', default="original_to_new_contig_name.tsv")
parser.add_argument("-v", "--verbose", help="increase output verbosity",
action="store_true")
@@ -70,7 +72,7 @@ def main():
sample = args.sample
fna_file = args.fna_file
out_fna = args.out_fna
- old2new_contig_name = "old_to_new_contig_name.tsv"
+ old2new_contig_name = args.contig_names_table
logging.info(f'Writting renamed fasta file in {out_fna}')
diff --git a/docs/output.md b/docs/output.md
index f5622a6..26ce39a 100644
--- a/docs/output.md
+++ b/docs/output.md
@@ -88,8 +88,7 @@ The `results/` directory contains a sub-directory for each step launched:
| `SAMPLE_NAME.fna` | unfiltered assembly (primary assembly) with renamed contigs. Contig names follow this pattern: `<sample_name>_c<contig_number>`. |
| `assembly_metric` | Contains metaQUAST quality control files of contigs. |
| `<assembly_tool>_SAMPLE_NAME/` | output files of the assembly tool. It contains logs and original fasta files of the assembly before renaming. |
-
-
+| `SAMPLE_NAME_original_to_new_contig_name.tsv` | Tabular file with two fields: original contig name from the assembly tool and the new contig name given by the pipeline. |
#### **02_assembly/02_2_deduplicated_reads**
diff --git a/modules/assembly.nf b/modules/assembly.nf
index 0b4d56f..8a7827c 100644
--- a/modules/assembly.nf
+++ b/modules/assembly.nf
@@ -157,10 +157,11 @@ process RENAME_CONTIGS {
output:
tuple val(meta), path("${meta.id}.fna"), emit: fna
+ path("${meta.id}_original_to_new_contig_name.tsv")
script:
"""
- rename_contigs.py --sample ${meta.id} --fna_file ${meta.id}.raw.fna --out_fna ${meta.id}.fna -v
+ rename_contigs.py --sample ${meta.id} --fna_file ${meta.id}.raw.fna --out_fna ${meta.id}.fna -v -t ${meta.id}_original_to_new_contig_name.tsv
"""
}
\ No newline at end of file
diff --git a/subworkflows/02_assembly.nf b/subworkflows/02_assembly.nf
index b53a0d3..ef0d29f 100644
--- a/subworkflows/02_assembly.nf
+++ b/subworkflows/02_assembly.nf
@@ -123,7 +123,9 @@ workflow STEP_02_ASSEMBLY {
}
}
- ch_assembly_renamed = RENAME_CONTIGS(ch_assembly)
+ RENAME_CONTIGS(ch_assembly)
+ ch_assembly_renamed = RENAME_CONTIGS.out.fna
+
ch_assembly_quast = ch_assembly_renamed
.map { meta, file -> file }
.collect()
--
GitLab
From 9fcc0021f6f6f18981ff847abdecada2fd09ee31 Mon Sep 17 00:00:00 2001
From: "maina.vienne" <maina.vienne@inrae.fr>
Date: Wed, 18 Jan 2023 17:22:07 +0100
Subject: [PATCH 180/194] update doc, remove warning
---
docs/usage.md | 23 +++++++----------------
1 file changed, 7 insertions(+), 16 deletions(-)
diff --git a/docs/usage.md b/docs/usage.md
index a0907e2..a62d587 100644
--- a/docs/usage.md
+++ b/docs/usage.md
@@ -303,30 +303,21 @@ No parameter available for this substep.
No parameters.
**WARNING 6:** `S04_STRUCTURAL_ANNOT` step depends on `S01_CLEAN_QC`, `S02_ASSEMBLY` and `S03_FILTERING` steps (if you use it). You need to use the mandatory files of these four steps to run `S04_STRUCTURAL_ANNOT`. See [II. Input files](usage.md#ii-input-files) and **WARNINGS 1 to 6**.
-<!--
-**WARNING 7:** if you haven't previously done `S03_FILTERING`, calculation time of `S04_STRUCTURAL_ANNOT` can be important. Some cluster queues have defined calculation time, you need to adapt the queue you use to your data.
-> For example, if you are on [genologin cluster](http://bioinfo.genotoul.fr/) and you haven't done the `S03_FILTERING` step, you can write a `nextflow.config` file in your working directory containing these lines:
-> ```bash
-> withName: PROKKA {
-> queue = 'unlimitq'
-> }
-> ```
-> This will launch the `Prokka` command line of step `04_STRUCTURAL_ANNOT` on a calculation queue (`unlimitq`) where the job can last more than 4 days (which is not the case for the usual `workq` queue). -->
#### **`S05_ALIGNMENT` step:**
-**WARNING 8:** `S05_ALIGNMENT` step depends on `S01_CLEAN_QC`, `S02_ASSEMBLY`, `S03_FILTERING` (if you use it) and `S04_STRUCTURAL_ANNOT` steps. You need to use the mandatory files of these five steps to run `S05_ALIGNMENT`. See [II. Input files](usage.md#ii-input-files) and **WARNINGS 1 to 8**.
+**WARNING 7:** `S05_ALIGNMENT` step depends on `S01_CLEAN_QC`, `S02_ASSEMBLY`, `S03_FILTERING` (if you use it) and `S04_STRUCTURAL_ANNOT` steps. You need to use the mandatory files of these five steps to run `S05_ALIGNMENT`. See [II. Input files](usage.md#ii-input-files) and **WARNINGS 1 to 7**.
* `--diamond_bank "<PATH>/bank.dmnd"`: path to diamond bank used to align protein sequence of genes. This bank must be previously built with [diamond makedb](https://github.com/bbuchfink/diamond/wiki). Default `""`.
Make sure that the version of the tools corresponds to the version of the bank specified in the parameter.
For example check the version of software in the yaml file and the singularity recipe in env/ repository and be sure that you specified the appropriate databank version.
-**WARNING 9:** You need to use a NCBI reference to have functional links in the output file _Quantifications_and_functional_annotations.tsv_ of `S06_FUNC_ANNOT` step
+**WARNING 8:** You need to use a NCBI reference to have functional links in the output file _Quantifications_and_functional_annotations.tsv_ of `S06_FUNC_ANNOT` step
#### **`S06_FUNC_ANNOT` step:**
-**WARNING 10:** `S06_FUNC_ANNOT` step depends on `S01_CLEAN_QC`, `S02_ASSEMBLY`, `S03_FILTERING` (if you use it), `S04_STRUCTURAL_ANNOT` and `S05_ALIGNMENT` steps. You need to use the mandatory files of these six steps to run `S06_FUNC_ANNOT`. See [II. Input files](usage.md#ii-input-files) and **WARNINGS 1 to 9**.
+**WARNING 9:** `S06_FUNC_ANNOT` step depends on `S01_CLEAN_QC`, `S02_ASSEMBLY`, `S03_FILTERING` (if you use it), `S04_STRUCTURAL_ANNOT` and `S05_ALIGNMENT` steps. You need to use the mandatory files of these six steps to run `S06_FUNC_ANNOT`. See [II. Input files](usage.md#ii-input-files) and **WARNINGS 1 to 8**.
* `--percentage_identity [number]`: corresponds to cd-hit-est -c option to indicate sequence percentage identity for clustering genes. Default: `0.95` corresponding to 95% of sequence identity. Use: `number` must be between 0 and 1, and use `.` when you want to use a float.
@@ -337,17 +328,17 @@ For example check the version of software in the yaml file and the singularity r
Make sure that the version of the tools corresponds to the version of the bank specified in the parameter.
For example check the version of software in the yaml file and the singularity recipe in env/ repository and be sure that you specified the appropriate databank version.
-**WARNING 11:** you need to use either `--eggnogmapper_db_download` or `--eggnog_mapper_db_dir`. If it is not the case, an error message will occur.
+**WARNING 10:** you need to use either `--eggnogmapper_db_download` or `--eggnog_mapper_db_dir`. If it is not the case, an error message will occur.
#### **`S07_TAXO_AFFI` step:**
-**WARNING 12:** `S07_TAXO_AFFI` step depends on `S01_CLEAN_QC`, `S02_ASSEMBLY`, `S03_FILTERING` (if you use it), `S04_STRUCTURAL_ANNOT` and `S05_ALIGNMENT` steps. You need to use the mandatory files of these six steps to run `S07_TAXO_AFFI`. See [II. Input files](usage.md#ii-input-files) and **WARNINGS 1 to 9**.
+**WARNING 11:** `S07_TAXO_AFFI` step depends on `S01_CLEAN_QC`, `S02_ASSEMBLY`, `S03_FILTERING` (if you use it), `S04_STRUCTURAL_ANNOT` and `S05_ALIGNMENT` steps. You need to use the mandatory files of these six steps to run `S07_TAXO_AFFI`. See [II. Input files](usage.md#ii-input-files) and **WARNINGS 1 to 8**.
* `--accession2taxid "<PATH>/prot.accession2taxid.FULL.gz"`: indicates the local path or FTP adress of the NCBI file `prot.accession2taxid.FULL.gz`. Default: `"ftp://ftp.ncbi.nih.gov/pub/taxonomy/accession2taxid/prot.accession2taxid.FULL.gz"`. The local file can be gzip or not.
* `--taxdump "<PATH>/new_taxdump.tar.gz"`: indicates the local path or the FTP adress of the NCBI file `taxdump.tar.gz`. Default `"ftp://ftp.ncbi.nih.gov/pub/taxonomy/new_taxdump/new_taxdump.tar.gz"`. The local file can be either a tar.gz archive or the extracted `new_taxdump` directory.
-**WARNING 13:** To have contigs and genes taxonomic affiliation your protein database used in the step 05_alignment has to come from ncbi and your taxdump and prot.accession2taxid files must be coherent, i.e. downloaded at the same time as the protein database used in 05_alignment step.
+**WARNING 12:** To have contigs and genes taxonomic affiliation your protein database used in the step 05_alignment has to come from ncbi and your taxdump and prot.accession2taxid files must be coherent, i.e. downloaded at the same time as the protein database used in 05_alignment step.
#### **`S08_binning` step:**
@@ -382,4 +373,4 @@ See the description of output files in [this part](output.md) of the documentati
> If you have an account on the [genologin cluster](http://bioinfo.genotoul.fr/) and you would like to familiarize yourself with metagWGS, see the tutorial available in the [use case documentation page](use_case.md). It allows the analysis of big test datasets with metagWGS. (WARNING: use_case documentation is not up-to-date, needs to be updated)
-**WARNING 16:** the test dataset in `metagwgs-test-datasets/small` used in [I. Basic Usage](usage.md#i-basic-usage) is a small test dataset which allows to test all steps but with few CPUs and memory.
+**WARNING 13:** the test dataset in `metagwgs-test-datasets/small` used in [I. Basic Usage](usage.md#i-basic-usage) is a small test dataset which allows to test all steps but with few CPUs and memory.
--
GitLab
From ec756bfbdab6d483a0cc2c5b954fa1da2d580f07 Mon Sep 17 00:00:00 2001
From: Maina Vienne <maina.vienne@inrae.fr>
Date: Wed, 18 Jan 2023 17:29:53 +0100
Subject: [PATCH 181/194] Update singularity image of metagWGS and binning
---
env/Singularity_recipe_binning | 9 ++++++---
env/metagWGS.yml | 26 ++++++++++++++------------
2 files changed, 20 insertions(+), 15 deletions(-)
diff --git a/env/Singularity_recipe_binning b/env/Singularity_recipe_binning
index 4221b25..91b90c6 100644
--- a/env/Singularity_recipe_binning
+++ b/env/Singularity_recipe_binning
@@ -11,17 +11,20 @@ apt-get update && apt-get install -y procps build-essential g++ && apt-get clean
/opt/conda/bin/mamba env create -f /binning.yml && /opt/conda/bin/conda clean -a
export PATH=/opt/conda/envs/binning/bin:$PATH
-git clone https://forgemia.inra.fr/jean.mainguy/binette.git
git clone --recursive https://github.com/chklovski/checkm2.git
pip install checkm2/
+export PATH=/opt/conda/envs/binning/bin/:/checkm2/bin/:$PATH
checkm2 database --download --path '.' || echo checkm2 database failed but it is probably ok
+git clone https://github.com/genotoul-bioinfo/Binette.git
+pip install Binette/
+
%environment
-export PATH=/opt/conda/envs/binning/bin/:/checkm2/bin/:/binette/binette/:$PATH
+export PATH=/opt/conda/envs/binning/bin/:/checkm2/bin/:$PATH
%runscript
"$@"
-
+
\ No newline at end of file
diff --git a/env/metagWGS.yml b/env/metagWGS.yml
index 6a0b710..463510d 100644
--- a/env/metagWGS.yml
+++ b/env/metagWGS.yml
@@ -4,29 +4,31 @@ channels:
- bioconda
- defaults
dependencies:
+ - barrnap=0.9
- bcbio-gff=0.6.9
- bwa-mem2=2.2.1
- cd-hit=4.8.1
- - cutadapt=4.1
+ - cutadapt=4.2
- diamond=2.0.15
- eggnog-mapper=2.1.9
- fastqc=0.11.9
+ - flye=2.9.1
- genometools-genometools=1.6.2
- - kaiju=1.9.0
+ - hifiasm_meta=hamtv0.3
+ - kaiju=1.9.2
- krona=2.8.1
- megahit=1.2.9
- minimap2=2.24
- - multiqc=1.13
- - pandas=1.1.5
- - prokka=1.14.6
- - python=3.7.12
+ - multiqc=1.14
+ - pandas=1.5.2
+ - plotly
+ - prodigal=2.6.3
+ - pyfastx
+ - python=3.10.8
- quast=5.2.0
- samtools=1.15.1
+ - scipy=1.9.3
- sickle-trim=1.33
- spades=3.15.5
- - subread=2.0.1
- - flye=2.9.1
- - hifiasm_meta=hamtv0.3
- - plotly
- - trnascan-se=2.0.11
- - pyfastx
+ - subread=2.0.3
+ - trnascan-se=2.0.11
\ No newline at end of file
--
GitLab
From 0f7b98f6a26e84c6ff078755bf9ff94ff185da64 Mon Sep 17 00:00:00 2001
From: "maina.vienne" <maina.vienne@inrae.fr>
Date: Wed, 18 Jan 2023 17:33:07 +0100
Subject: [PATCH 182/194] Fix for long read feature Count with option -L
---
modules/feature_counts.nf | 31 ++++++++++++++++---------------
1 file changed, 16 insertions(+), 15 deletions(-)
diff --git a/modules/feature_counts.nf b/modules/feature_counts.nf
index 63eb79a..79413f0 100644
--- a/modules/feature_counts.nf
+++ b/modules/feature_counts.nf
@@ -1,23 +1,24 @@
// Quantification of reads on each gene in each sample.
process FEATURE_COUNTS {
- tag "${meta.id}"
- label 'QUANTIFICATION'
- publishDir "${params.outdir}/06_func_annot/06_2_quantification", mode: 'copy', pattern: "${meta.id}.featureCounts*"
+ tag "${meta.id}"
+ label 'QUANTIFICATION'
+ publishDir "${params.outdir}/06_func_annot/06_2_quantification", mode: 'copy', pattern: "${meta.id}.featureCounts*"
- input:
- tuple val(meta), file(gff_prokka), file(bam), file(bam_index)
+ input:
+ tuple val(meta), file(gff_prokka), file(bam), file(bam_index)
- output:
- path "${meta.id}.featureCounts.tsv", emit: count_table
- path "${meta.id}.featureCounts.tsv.summary", emit: summary
- path "${meta.id}.featureCounts.stdout"
- path "v_featurecounts.txt", emit: v_featurecounts
+ output:
+ path "${meta.id}.featureCounts.tsv", emit: count_table
+ path "${meta.id}.featureCounts.tsv.summary", emit: summary
+ path "${meta.id}.featureCounts.stdout"
+ path "v_featurecounts.txt", emit: v_featurecounts
- script:
- """
- featureCounts -T ${task.cpus} -p -O -t gene -g ID -a ${gff_prokka} -o ${meta.id}.featureCounts.tsv ${bam} &> ${meta.id}.featureCounts.stdout
- featureCounts -v &> v_featurecounts.txt
- """
+ script:
+ if (meta.type=="SR"){ option = "-p --countReadPairs" } else { option = "-L" }
+ """
+ featureCounts -T ${task.cpus} $option -O -t gene -g ID -a ${gff_prokka} -o ${meta.id}.featureCounts.tsv ${bam} &> ${meta.id}.featureCounts.stdout
+ featureCounts -v &> v_featurecounts.txt
+ """
}
// Create table with sum of reads for each global cluster of genes in each sample.
--
GitLab
From 7e63686252675775af6cf7aa7e11a9a712e97dfa Mon Sep 17 00:00:00 2001
From: "maina.vienne" <maina.vienne@inrae.fr>
Date: Wed, 18 Jan 2023 17:42:59 +0100
Subject: [PATCH 183/194] Small fix for reproductible output for metaquast
---
modules/metaquast.nf | 3 +--
subworkflows/02_assembly.nf | 8 ++++----
subworkflows/03_filtering.nf | 2 +-
3 files changed, 6 insertions(+), 7 deletions(-)
diff --git a/modules/metaquast.nf b/modules/metaquast.nf
index bc20ad7..a143cf3 100644
--- a/modules/metaquast.nf
+++ b/modules/metaquast.nf
@@ -7,13 +7,12 @@ process QUAST {
val outdir
output:
- path("assembly_metric/"), emit: all
+ path("assembly_metric/*"), emit: all
path("assembly_metric/report.tsv"), emit: report
path("v_quast.txt"), emit: v_quast
script:
"""
- touch report.tsv
metaquast.py --threads ${task.cpus} --rna-finding --max-ref-number 0 --min-contig 0 ${assemblies} -o assembly_metric/
quast -v &> v_quast.txt
diff --git a/subworkflows/02_assembly.nf b/subworkflows/02_assembly.nf
index ef0d29f..12f75cd 100644
--- a/subworkflows/02_assembly.nf
+++ b/subworkflows/02_assembly.nf
@@ -39,15 +39,15 @@ workflow STEP_02_ASSEMBLY {
flowcell:meta.flowcell.join("_"),
group:meta.group.unique().join(),
assembly:meta.assembly.unique().join(),
- type:meta.type.unique().join()], fastq.flatten() ]
+ type:meta.type.unique().join()], fastq.flatten().sort{ it.baseName } ]
multiple: fastq.size() > 1
return [[id:meta.sample.unique().join(),
sample:meta.sample.unique().join(),
flowcell:meta.flowcell.join("_"),
group:meta.group.unique().join(),
assembly:meta.assembly.unique().join(),
- type:meta.type.unique().join()], fastq.flatten() ]
- }
+ type:meta.type.unique().join()], fastq.flatten().sort{ it.baseName } ]
+ }
MERGE_FASTQ (ch_reads_flowcell.multiple)
.reads
@@ -128,7 +128,7 @@ workflow STEP_02_ASSEMBLY {
ch_assembly_quast = ch_assembly_renamed
.map { meta, file -> file }
- .collect()
+ .collect(sort:{it.baseName})
if ( params.type.toUpperCase() == "HIFI" ) {
CIRCULAR_CONTIGS(assembly_tool, ch_assembly_renamed, ch_infos)
diff --git a/subworkflows/03_filtering.nf b/subworkflows/03_filtering.nf
index bf92a0b..810b5e6 100644
--- a/subworkflows/03_filtering.nf
+++ b/subworkflows/03_filtering.nf
@@ -77,7 +77,7 @@ workflow STEP_03_ASSEMBLY_FILTER {
ch_merged_selected_all = ch_merged_selected
.map { meta, file -> file }
- .collect()
+ .collect(sort:{it.baseName})
QUAST( ch_merged_selected_all, "${filtering_outdir}/03_1_filtered_assembly/" )
ch_quast_report = QUAST.out.report
--
GitLab
From 2a68704611a44936f1b8e45bf2198c35d0d33510 Mon Sep 17 00:00:00 2001
From: "maina.vienne" <maina.vienne@inrae.fr>
Date: Thu, 19 Jan 2023 12:13:47 +0100
Subject: [PATCH 184/194] Retrieve circular contigs for HiFi on step 02
---
bin/retrieve_circular_contigs.py | 42 ++++++++++++++++++++---------
modules/assembly.nf | 46 +-------------------------------
modules/circular_contigs.nf | 34 +++++++++++++++++++++++
modules/rename_contigs.nf | 20 ++++++++++++++
subworkflows/02_assembly.nf | 24 ++++++++++-------
5 files changed, 98 insertions(+), 68 deletions(-)
create mode 100644 modules/circular_contigs.nf
create mode 100644 modules/rename_contigs.nf
diff --git a/bin/retrieve_circular_contigs.py b/bin/retrieve_circular_contigs.py
index 565190f..7c18ab4 100755
--- a/bin/retrieve_circular_contigs.py
+++ b/bin/retrieve_circular_contigs.py
@@ -4,10 +4,11 @@
from Bio import SeqIO
import pandas as pd
import argparse
+import os
## Functions
-def parse_metaflye(input_fasta, info_file, circular_fasta):
+def parse_metaflye(input_fasta, info_file, outdir):
"""
metaflye fasta contains no information.
This is contained in the assembly_info.txt file.
@@ -16,19 +17,31 @@ def parse_metaflye(input_fasta, info_file, circular_fasta):
# this file may have changed symbols for yes, possibilities include a Y or +
circular_contigs = list(df[df['circ.'].isin(['Y', '+'])]["#seq_name"])
- with open(circular_fasta, 'wt') as fh_out:
- for rec in SeqIO.parse(input_fasta, "fasta"):
- if rec.id in circular_contigs:
- fh_out.write(rec.format("fasta"))
+ for count, rec in enumerate(SeqIO.parse(input_fasta, "fasta")):
+ if rec.id in circular_contigs:
+ outfile = os.path.join(outdir, f"bin_{count}.fa")
+ with open(outfile, 'wt') as outfl:
+ outfl.write(rec.format("fasta"))
-def parse_hifiasm(input_fasta, circular_fasta):
+ # with open(circular_fasta, 'wt') as fh_out:
+ # for rec in SeqIO.parse(input_fasta, "fasta"):
+ # if rec.id in circular_contigs:
+ # fh_out.write(rec.format("fasta"))
+
+def parse_hifiasm(input_fasta, outdir):
"""
hifiasm-meta circular contig names will have an "c" suffix.
"""
- with open(circular_fasta, 'wt') as fh_out:
- for rec in SeqIO.parse(input_fasta, "fasta"):
- if rec.id.endswith('c'):
- fh_out.write(rec.format("fasta"))
+ for count, rec in enumerate(SeqIO.parse(input_fasta, "fasta")):
+ if rec.description.endswith('c'):
+ outfile = os.path.join(outdir, f"bin_{count}.fa")
+ with open(outfile, 'wt') as outfl:
+ outfl.write(rec.format("fasta"))
+
+ # with open(circular_fasta, 'wt') as fh_out:
+ # for rec in SeqIO.parse(input_fasta, "fasta"):
+ # if rec.id.endswith('c'):
+ # fh_out.write(rec.format("fasta"))
def main():
# Manage parameters
@@ -42,19 +55,22 @@ def main():
group_input_otu_table.add_argument('-i','--info-file', default=None, help="The path of metaflye asembly info file.")
# output
group_output = parser.add_argument_group( 'Outputs' )
- group_output.add_argument('-o','--output-fasta', default='circular_contigs.fna', help="The path of circular contigs fasta file. [Default: %(default)s]" )
+ group_output.add_argument('-o','--outdir', default='circular_contigs', help="The path of circular contigs directory. [Default: %(default)s]" )
args = parser.parse_args()
+ outdir = args.outdir
+ os.makedirs(outdir, exist_ok=True)
+
# Check for inputs
if args.assembler == "metaflye":
if args.info_file is None:
parser.error("\n\n#ERROR : --info-file is required with metaflye assembler.")
else:
- parse_metaflye(args.input_fasta, args.info_file, args.output_fasta)
+ parse_metaflye(args.input_fasta, args.info_file, outdir)
elif args.assembler == "hifiasm-meta":
- parse_hifiasm(args.input_fasta, args.output_fasta)
+ parse_hifiasm(args.input_fasta, outdir)
if __name__ == '__main__':
diff --git a/modules/assembly.nf b/modules/assembly.nf
index 8a7827c..4b7f87f 100644
--- a/modules/assembly.nf
+++ b/modules/assembly.nf
@@ -106,7 +106,7 @@ process METAFLYE {
output:
tuple val(meta), path("metaflye_${meta.id}/${meta.id}.contigs.fa"), emit: assembly
tuple val(meta.id), path("metaflye_${meta.id}/${meta.id}.log"), path("metaflye_${meta.id}/${meta.id}.params.json"), emit: report
- tuple val(meta.id), path("metaflye_${meta.id}/assembly_info.txt"), emit: infos
+ tuple val(meta), path("metaflye_${meta.id}/assembly_info.txt"), emit: infos
path "v_metaflye.txt", emit: v_metaflye
script:
@@ -120,48 +120,4 @@ process METAFLYE {
flye --version &> v_metaflye.txt
"""
-}
-
-process CIRCULAR_CONTIGS {
- tag "${meta.id}"
- publishDir "${params.outdir}/02_assembly/02_1_primary_assembly", mode: 'copy'
- label 'PYTHON'
-
- input:
- val(assembler)
- tuple val(meta), path("${meta.id}.fna")
- path(infos)
-
- output:
- tuple val(meta), path("${meta.id}.circular.contigs.fna"), emit: circular
-
- script:
- """
- if [ $assembler == 'metaflye' ]
- then
- retrieve_circular_contigs.py -a metaflye -f ${meta.id}.fna -i $infos -o ${meta.id}.circular.contigs.fna
- elif [ $assembler == 'hifiasm-meta' ]
- then
- retrieve_circular_contigs.py -a hifiasm-meta -f ${meta.id}.fna -o ${meta.id}.circular.contigs.fna
- fi
- """
-}
-
-process RENAME_CONTIGS {
- tag "${meta.id}"
- publishDir "${params.outdir}/02_assembly/02_1_primary_assembly", mode: 'copy'
- label 'PYTHON'
-
- input:
- tuple val(meta), path("${meta.id}.raw.fna")
-
- output:
- tuple val(meta), path("${meta.id}.fna"), emit: fna
- path("${meta.id}_original_to_new_contig_name.tsv")
-
- script:
- """
- rename_contigs.py --sample ${meta.id} --fna_file ${meta.id}.raw.fna --out_fna ${meta.id}.fna -v -t ${meta.id}_original_to_new_contig_name.tsv
-
- """
}
\ No newline at end of file
diff --git a/modules/circular_contigs.nf b/modules/circular_contigs.nf
new file mode 100644
index 0000000..b7bae04
--- /dev/null
+++ b/modules/circular_contigs.nf
@@ -0,0 +1,34 @@
+process CIRCULAR_CONTIGS_METAFLYE {
+ tag "${meta.id}"
+ publishDir "${params.outdir}/02_assembly/02_1_primary_assembly", mode: 'copy'
+ label 'PYTHON'
+
+ input:
+ tuple val(meta), path("${meta.id}.fna"), path(infos)
+
+ output:
+ tuple val(meta), path("circular_contigs/"), emit: circular
+
+ script:
+ """
+ retrieve_circular_contigs.py -a 'metaflye' -f ${meta.id}.fna -i ${infos} -o circular_contigs/
+ """
+}
+
+
+process CIRCULAR_CONTIGS_HIFIASM {
+ tag "${meta.id}"
+ publishDir "${params.outdir}/02_assembly/02_1_primary_assembly", mode: 'copy'
+ label 'PYTHON'
+
+ input:
+ tuple val(meta), path("${meta.id}.fna")
+
+ output:
+ tuple val(meta), path("circular_contigs/"), emit: circular
+
+ script:
+ """
+ retrieve_circular_contigs.py -a 'hifiasm-meta' -f ${meta.id}.fna -o circular_contigs/
+ """
+}
\ No newline at end of file
diff --git a/modules/rename_contigs.nf b/modules/rename_contigs.nf
new file mode 100644
index 0000000..d6b67a9
--- /dev/null
+++ b/modules/rename_contigs.nf
@@ -0,0 +1,20 @@
+process RENAME_CONTIGS {
+ tag "${meta.id}"
+ publishDir "${params.outdir}/02_assembly/02_1_primary_assembly", mode: 'copy'
+ label 'PYTHON'
+
+ input:
+ tuple val(meta), path("${meta.id}.raw.fna")
+
+
+
+ output:
+ tuple val(meta), path("${meta.id}.fna"), emit: fna
+ path("${meta.id}_original_to_new_contig_name.tsv")
+
+ script:
+ """
+ rename_contigs.py --sample ${meta.id} --fna_file ${meta.id}.raw.fna --out_fna ${meta.id}.fna -v -t ${meta.id}_original_to_new_contig_name.tsv
+
+ """
+}
\ No newline at end of file
diff --git a/subworkflows/02_assembly.nf b/subworkflows/02_assembly.nf
index 12f75cd..94e1771 100644
--- a/subworkflows/02_assembly.nf
+++ b/subworkflows/02_assembly.nf
@@ -1,4 +1,6 @@
-include { METASPADES; MEGAHIT; HIFIASM_META; METAFLYE; CIRCULAR_CONTIGS; RENAME_CONTIGS } from '../modules/assembly'
+include { METASPADES; MEGAHIT; HIFIASM_META; METAFLYE } from '../modules/assembly'
+include { CIRCULAR_CONTIGS_METAFLYE; CIRCULAR_CONTIGS_HIFIASM } from '../modules/circular_contigs.nf'
+include { RENAME_CONTIGS } from '../modules/rename_contigs.nf'
include { QUAST as ASSEMBLY_QUAST} from '../modules/metaquast'
include { READS_DEDUPLICATION } from '../modules/reads_deduplication'
include { MINIMAP2 } from '../modules/read_alignment'
@@ -19,7 +21,8 @@ workflow STEP_02_ASSEMBLY {
ch_bwa_mem_v = Channel.empty()
ch_minimap2_v = Channel.empty()
ch_samtools_v = Channel.empty()
-
+ ch_infos_metaflye = Channel.empty()
+ ch_circular = Channel.empty()
ch_assembly = assembly
ch_reads = reads
@@ -47,7 +50,7 @@ workflow STEP_02_ASSEMBLY {
group:meta.group.unique().join(),
assembly:meta.assembly.unique().join(),
type:meta.type.unique().join()], fastq.flatten().sort{ it.baseName } ]
- }
+ }
MERGE_FASTQ (ch_reads_flowcell.multiple)
.reads
@@ -99,7 +102,6 @@ workflow STEP_02_ASSEMBLY {
}
if (!has_assembly){
- ch_circular = Channel.empty()
if (assembly_tool == 'metaspades') {
METASPADES(ch_reads_assembly)
ch_assembly = METASPADES.out.assembly
@@ -109,14 +111,13 @@ workflow STEP_02_ASSEMBLY {
ch_assembly = MEGAHIT.out.assembly
ch_assembler_v = MEGAHIT.out.v_megahit
} else if (assembly_tool == 'hifiasm-meta') {
- ch_infos = Channel.empty()
HIFIASM_META(ch_reads_assembly)
ch_assembly = HIFIASM_META.out.assembly
ch_assembler_v = HIFIASM_META.out.v_hifiasm_meta
} else if (assembly_tool == 'metaflye') {
METAFLYE(ch_reads_assembly)
ch_assembly = METAFLYE.out.assembly
- ch_infos = METAFLYE.out.infos
+ ch_infos_metaflye = METAFLYE.out.infos
ch_assembler_v = METAFLYE.out.v_metaflye
} else {
exit 1, "Invalid assembly parameter: ${assembly_tool}"
@@ -130,10 +131,13 @@ workflow STEP_02_ASSEMBLY {
.map { meta, file -> file }
.collect(sort:{it.baseName})
- if ( params.type.toUpperCase() == "HIFI" ) {
- CIRCULAR_CONTIGS(assembly_tool, ch_assembly_renamed, ch_infos)
- ch_circular = CIRCULAR_CONTIGS.out.circular
-
+ if (assembly_tool == 'hifiasm-meta') {
+ CIRCULAR_CONTIGS_HIFIASM(ch_assembly_renamed)
+ ch_circular = CIRCULAR_CONTIGS_HIFIASM.out.circular
+ } else if (assembly_tool == 'metaflye') {
+ ch_assembly_metaflye_infos = ch_assembly_renamed.join(ch_infos_metaflye)
+ CIRCULAR_CONTIGS_METAFLYE(ch_assembly_metaflye_infos)
+ ch_circular = CIRCULAR_CONTIGS_METAFLYE.out.circular
}
ASSEMBLY_QUAST( ch_assembly_quast,"02_assembly/02_1_primary_assembly/")
--
GitLab
From 2adbe555696479ddc3f46a3d45aabdf22e1a0740 Mon Sep 17 00:00:00 2001
From: "maina.vienne" <maina.vienne@inrae.fr>
Date: Thu, 19 Jan 2023 12:15:56 +0100
Subject: [PATCH 185/194] Update poster name
---
communication/Poster_ECCB2022.pdf | Bin 0 -> 836151 bytes
...m2020_poster_9.pdf => Poster_JOBIM2020.pdf} | Bin
...Jobim_metagWGS.pdf => Poster_JOBIM2022.pdf} | Bin
3 files changed, 0 insertions(+), 0 deletions(-)
create mode 100755 communication/Poster_ECCB2022.pdf
rename communication/{jobim2020_poster_9.pdf => Poster_JOBIM2020.pdf} (100%)
rename communication/{Poster_Jobim_metagWGS.pdf => Poster_JOBIM2022.pdf} (100%)
diff --git a/communication/Poster_ECCB2022.pdf b/communication/Poster_ECCB2022.pdf
new file mode 100755
index 0000000000000000000000000000000000000000..c9803fbc93974f68a53c92df71b4a47e6e298721
GIT binary patch
literal 836151
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diff --git a/communication/jobim2020_poster_9.pdf b/communication/Poster_JOBIM2020.pdf
similarity index 100%
rename from communication/jobim2020_poster_9.pdf
rename to communication/Poster_JOBIM2020.pdf
diff --git a/communication/Poster_Jobim_metagWGS.pdf b/communication/Poster_JOBIM2022.pdf
similarity index 100%
rename from communication/Poster_Jobim_metagWGS.pdf
rename to communication/Poster_JOBIM2022.pdf
--
GitLab
From 691adf488f65386346da693534d530fa37b62919 Mon Sep 17 00:00:00 2001
From: "maina.vienne" <maina.vienne@inrae.fr>
Date: Fri, 20 Jan 2023 16:31:04 +0100
Subject: [PATCH 186/194] Use circular contigs in binning
---
main.nf | 7 +++---
modules/binning.nf | 18 ++++++++------
subworkflows/08_binning.nf | 49 +++++++++++++++++++++++++-------------
3 files changed, 47 insertions(+), 27 deletions(-)
diff --git a/main.nf b/main.nf
index b42462b..51f06c6 100644
--- a/main.nf
+++ b/main.nf
@@ -335,6 +335,7 @@ workflow {
ch_software_versions_S07 = Channel.empty()
ch_software_versions_S08 = Channel.empty()
ch_software_versions_total = Channel.empty()
+ ch_circular = Channel.empty()
ch_multiqc_config = file(params.multiqc_config, checkIfExists: true)
@@ -376,9 +377,7 @@ workflow {
ch_quast_before_filter_report = S02_ASSEMBLY.out.assembly_report
ch_software_versions_S02 = S02_ASSEMBLY.out.software_versions
- if (params.type.toUpperCase() == 'HIFI') {
- ch_circular = S02_ASSEMBLY.out.circular
- }
+ ch_circular = S02_ASSEMBLY.out.circular
}
if ( !params.stop_at_clean && !params.stop_at_assembly && !params.skip_filtering ) {
@@ -451,7 +450,7 @@ workflow {
if ( !params.stop_at_clean && !params.stop_at_assembly && !params.stop_at_filtering && !params.stop_at_structural_annot && !params.skip_binning ) {
ch_quast=ch_quast_after_filter_report.ifEmpty(ch_quast_before_filter_report)
- S08_BINNING( ch_reads, ch_assembly, ch_bam, ch_gtbdtk_db, ch_quast )
+ S08_BINNING( ch_reads, ch_assembly, ch_bam, ch_gtbdtk_db, ch_quast, ch_circular)
ch_bins_abundances_report = S08_BINNING.out.bins_abundances_report
ch_bins_stats_report = S08_BINNING.out.bins_stats_report
diff --git a/modules/binning.nf b/modules/binning.nf
index 9cc6879..6d90b0f 100644
--- a/modules/binning.nf
+++ b/modules/binning.nf
@@ -124,13 +124,13 @@ process BINETTE {
val max_contamination
output:
- tuple val(meta), path("bin_refinement/final_bins/*"), emit: bins
+ tuple val(meta), path("bin_refinement/final_bins/*"), emit: bins, optional: true
tuple val(meta), path('bins_stats.tsv'), emit: checkm_stats
path "v_binette.txt", emit: v_binette
script:
"""
- binette.py -v -m 0 -t ${task.cpus} -d ${bins.join(' ')} -c ${contigs} -o bin_refinement/
+ binette -v -m ${min_completeness} -t ${task.cpus} -d ${bins.join(' ')} -c ${contigs} -o bin_refinement/
sed -i 's/size/Size/' bin_refinement/final_bins_quality_reports.tsv
sed -i 's/^/${meta.id}_bin_/' bin_refinement/final_bins_quality_reports.tsv
@@ -139,13 +139,17 @@ process BINETTE {
cd bin_refinement/final_bins
- for filename in *fa;
- do
- mv \$filename "${meta.id}_\$filename"
- done
+ if [[ \$(ls ./) ]] #check if directoy not empty
+ then
+ for filename in *fa;
+ do
+ mv \$filename "${meta.id}_\$filename"
+ done
+ fi
cd ../..
- touch v_binette.txt
+
+ binette --version > v_binette.txt
"""
}
diff --git a/subworkflows/08_binning.nf b/subworkflows/08_binning.nf
index df6ca95..eceb6a0 100644
--- a/subworkflows/08_binning.nf
+++ b/subworkflows/08_binning.nf
@@ -16,6 +16,7 @@ workflow STEP_08_BINNING {
bam
gtdbtk_db
quast
+ circular
main:
@@ -136,11 +137,11 @@ workflow STEP_08_BINNING {
ch_concoct_bins = CONCOCT.out.bins.filter{ t -> t[1].list().size()}
ch_concoct_v = CONCOCT.out.v_concoct
-
+ ch_circular = circular.filter{ t -> t[1].list().size()}
//////////////////
//// BIN REFINEMENT
//////////////////
- ch_bins_set = ch_metabat_bins.mix(ch_concoct_bins, ch_maxbin_bins)
+ ch_bins_set = ch_metabat_bins.mix(ch_concoct_bins, ch_maxbin_bins, ch_circular)
.groupTuple(by: [0])
.branch{ meta, bins ->
multiple: bins.size() > 1
@@ -156,12 +157,23 @@ workflow STEP_08_BINNING {
ch_binette_bins = Channel.empty()
ch_binette_stats = Channel.empty()
ch_bins_assembly = Channel.empty()
+ ch_drep_fna = Channel.empty()
+ ch_binette_bins_filter = Channel.empty()
+ ch_binette_stats_filter = Channel.empty()
+ ch_bins_drep = Channel.empty()
+ ch_bam_bins = Channel.empty()
+ ch_reads_fna = Channel.empty()
+ ch_gtdbtk_affi = Channel.empty()
+ ch_drep_stats = Channel.empty()
ch_bins_assembly = ch_bins_set.multiple.join(ch_assembly)
BINETTE(ch_bins_assembly, params.min_completeness, params.max_contamination)
ch_binette_bins = BINETTE.out.bins
- ch_binette_stats = BINETTE.out.checkm_stats
+
+ ch_binette_stats = BINETTE.out.checkm_stats.join(ch_binette_bins)
+ .map { meta, stats, bins ->
+ [ meta, stats ] }
ch_bins_assembly = ch_binette_bins.join(ch_assembly)
@@ -183,16 +195,20 @@ workflow STEP_08_BINNING {
///// DEREPLICATION BIN
////////////////////////
- ch_bins_drep = Channel.empty()
- ch_binette_bins = ch_binette_bins.map { meta, file -> file}
- .collect()
+ ch_bins_filter=ch_binette_bins.filter{ it[1].getClass() == java.util.ArrayList }
+ ch_binette_bins_filter = ch_bins_filter.map { meta, file -> file}
+ .collect()
- ch_binette_stats = ch_binette_stats.map { meta, file -> file}
- .collect()
-
- DREP(ch_binette_stats, ch_binette_bins, params.drep_threshold)
+ ch_binette_stats_filter = ch_binette_stats.join(ch_bins_filter)
+ .map { meta, stats, bins -> stats }
+ .collect()
+
+
+ DREP(ch_binette_stats_filter, ch_binette_bins_filter, params.drep_threshold)
ch_bins_drep = DREP.out.drep_bins_folder
ch_dRep_v = DREP.out.v_dRep
+ ch_drep_fna = DREP.out.fna
+ ch_drep_stats = DREP.out.output_drep_stats
///////////////////////////////
///// TAXONOMIC AFFILIATION BIN
@@ -200,35 +216,36 @@ workflow STEP_08_BINNING {
GTDBTK(ch_bins_drep, gtdbtk_db)
ch_gtdbtk_v = GTDBTK.out.v_gtdbtk
+ ch_gtdbtk_affi = GTDBTK.out.gtdbtk_affiliations_predictions
/////////////////////////////
////GENOMES ABUNDANCES
/////////////////////////////
- ch_reads_fna = reads.combine(DREP.out.fna)
+ ch_reads_fna = reads.combine(ch_drep_fna)
.map { meta, reads, bins -> [meta, bins, reads] }
if (params.type == 'SR') {
BWA_MEM(ch_reads_fna, "08_binning/08_4_mapping_on_final_bins/mapping")
- ch_bam = BWA_MEM.out.bam
+ ch_bam_bins = BWA_MEM.out.bam
ch_bwa_mem_v = BWA_MEM.out.v_bwa_mem2
ch_samtools_v = BWA_MEM.out.v_samtools
} else {
MINIMAP2(ch_reads_fna, "08_binning/08_4_mapping_on_final_bins/mapping")
- ch_bam = MINIMAP2.out.bam
+ ch_bam_bins = MINIMAP2.out.bam
ch_minimap2_v = MINIMAP2.out.v_minimap2
ch_samtools_v = MINIMAP2.out.v_samtools
}
ch_collect_coverages = Channel.empty()
ch_collect_flagstats = Channel.empty()
- GET_ALIGNMENT_METRICS(ch_bam, "08_binning/08_4_mapping_on_final_bins/stats")
+ GET_ALIGNMENT_METRICS(ch_bam_bins, "08_binning/08_4_mapping_on_final_bins/stats")
ch_collect_coverages = GET_ALIGNMENT_METRICS.out.sam_coverage.map {id, file -> file}
.collect()
ch_collect_flagstats = GET_ALIGNMENT_METRICS.out.sam_flagstat.collect()
- GENOMES_ABUNDANCES_PER_SAMPLE(ch_collect_coverages, ch_collect_flagstats, \
- ch_bins_drep, DREP.out.output_drep_stats, GTDBTK.out.gtdbtk_affiliations_predictions,
+ GENOMES_ABUNDANCES_PER_SAMPLE(ch_collect_coverages, ch_collect_flagstats,
+ ch_bins_drep, ch_drep_stats , ch_gtdbtk_affi ,
ch_heatmap_header_multiqc, ch_table_header_multiqc)
ch_bins_abundances_report = GENOMES_ABUNDANCES_PER_SAMPLE.out.report
--
GitLab
From 0065892cd8d9e63ad481a9ab55ca148437c2c43d Mon Sep 17 00:00:00 2001
From: "maina.vienne" <maina.vienne@inrae.fr>
Date: Fri, 20 Jan 2023 16:32:24 +0100
Subject: [PATCH 187/194] Update doc
---
bin/scrape_software_versions.py | 4 +-
docs/installation.md | 2 +-
docs/output.md | 85 +++++++++++++++++----------------
docs/usage.md | 2 +-
main.nf | 7 +--
nextflow.config | 3 --
6 files changed, 49 insertions(+), 54 deletions(-)
diff --git a/bin/scrape_software_versions.py b/bin/scrape_software_versions.py
index 4279474..199170a 100755
--- a/bin/scrape_software_versions.py
+++ b/bin/scrape_software_versions.py
@@ -30,7 +30,7 @@ regexes = {
'Maxbin': ['v_maxbin.txt', r"MaxBin (\S+)"],
'Metabat2': ['v_metabat2.txt', r"version (\S+)"],
'CheckM2': ['v_checkm2.txt', r"(\S+)"],
- 'Metawrap': ['v_metawrap.txt', r"metawrap (\S+)"],
+ 'Binette': ['v_binette.txt', r"(\S+)"],
'GTDBTK': ['v_gtdbtk.txt', r"...::: GTDB-Tk v(\S+)"],
'dRep': ['v_dRep.txt', r"version (\S+)"],
'tRNAscan-SE': ['v_tRNAscan.txt', r"tRNAscan-SE (\S+)"],
@@ -63,7 +63,7 @@ results['Concoct'] = '<span style="color:#999999;\">N/A</span>'
results['Metabat2'] = '<span style="color:#999999;\">N/A</span>'
results['Maxbin'] = '<span style="color:#999999;\">N/A</span>'
results['CheckM2'] = '<span style="color:#999999;\">N/A</span>'
-results['Metawrap'] = '<span style="color:#999999;\">N/A</span>'
+results['Binette'] = '<span style="color:#999999;\">N/A</span>'
results['dRep'] = '<span style="color:#999999;\">N/A</span>'
results['GTDBTK'] = '<span style="color:#999999;\">N/A</span>'
results['MultiQC'] = '<span style="color:#999999;\">N/A</span>'
diff --git a/docs/installation.md b/docs/installation.md
index f2d67cc..13a473c 100644
--- a/docs/installation.md
+++ b/docs/installation.md
@@ -57,7 +57,7 @@ singularity pull binning.sif oras://registry.forgemia.inra.fr/genotoul-bioinfo/m
two files (`metagwgs.sif` and `binning.sif`) must have been downloaded.
-**NOTE: if you are using the devel branch, you must use `oras://registry.forgemia.inra.fr/genotoul-bioinfo/metagwgs/binning_devel:latest` and `oras://registry.forgemia.inra.fr/genotoul-bioinfo/metagwgs/metagwgs_devel:latest`**
+**NOTE: if you are using the devel branch, you must use ` singularity pull binning.sif oras://registry.forgemia.inra.fr/genotoul-bioinfo/metagwgs/binning_devel:latest` and ` singularity pull metagwgs.sif oras://registry.forgemia.inra.fr/genotoul-bioinfo/metagwgs/metagwgs_devel:latest`**
### Solution 2: build the two containers.
diff --git a/docs/output.md b/docs/output.md
index 26ce39a..f32070e 100644
--- a/docs/output.md
+++ b/docs/output.md
@@ -89,6 +89,7 @@ The `results/` directory contains a sub-directory for each step launched:
| `assembly_metric` | Contains metaQUAST quality control files of contigs. |
| `<assembly_tool>_SAMPLE_NAME/` | output files of the assembly tool. It contains logs and original fasta files of the assembly before renaming. |
| `SAMPLE_NAME_original_to_new_contig_name.tsv` | Tabular file with two fields: original contig name from the assembly tool and the new contig name given by the pipeline. |
+| `circular_contigs/` | only for HiFi:
#### **02_assembly/02_2_deduplicated_reads**
@@ -161,36 +162,36 @@ The `results/` directory contains a sub-directory for each step launched:
| File | Description |
| ----------------------- | --------------------------------------- |
-| `SAMPLE_NAME.cd-hit-est.[cd-hit percentage identity].fasta` | Nucleotide sequences of representatives genes ("intra-sample clusters") generated by cd-hit-est with [cd-hit percentage identity] percentage identity. |
-| `SAMPLE_NAME.cd-hit-est.[cd-hit percentage identity].fasta.clstr` | Text file of list of intra-sample clusters generated by cd-hit-est with [cd-hit percentage identity] percentage identity. |
+| `SAMPLE_NAME.cd-hit-est.[cd-hit percentage identity].fasta` | Nucleotide sequences of representatives genes ("intra-sample clusters") generated by cd-hit-est with [cd-hit percentage identity] percentage identity. |
+| `SAMPLE_NAME.cd-hit-est.[cd-hit percentage identity].fasta.clstr` | Text file of list of intra-sample clusters generated by cd-hit-est with [cd-hit percentage identity] percentage identity. |
| `SAMPLE_NAME.cd-hit-est.[cd-hit percentage identity].table_cluster_contigs.txt` | Correspondance table of intra-sample clusters and initial genes. One line = one correspondance between an intra-sample cluster (first column) and an initial gene (second column). |
-| `All-cd-hit-est.[cd-hit percentage identity].fasta` | Nucleotide sequences of global representatives genes ("inter-sample clusters") generated by cd-hit-est with [cd-hit percentage identity] percentage identity. |
-| `All-cd-hit-est.[cd-hit percentage identity].fasta.clstr` | Text file of list of inter-sample clusters generated by cd-hit-est with [cd-hit percentage identity] percentage identity. |
-| `table_clstr.txt` | Correspondance table of inter-sample clusters and intra-sample clusters. One line = one correspondance between an inter-sample cluster (first column) and an intra-sample cluster (second column). |
+| `All-cd-hit-est.[cd-hit percentage identity].fasta` | Nucleotide sequences of global representatives genes ("inter-sample clusters") generated by cd-hit-est with [cd-hit percentage identity] percentage identity. |
+| `All-cd-hit-est.[cd-hit percentage identity].fasta.clstr` | Text file of list of inter-sample clusters generated by cd-hit-est with [cd-hit percentage identity] percentage identity. |
+| `table_clstr.txt` | Correspondance table of inter-sample clusters and intra-sample clusters. One line = one correspondance between an inter-sample cluster (first column) and an intra-sample cluster (second column). |
#### **06_func_annot/06_2_quantification/**
| File | Description |
| ----------------------- | --------------------------------------- |
-| `SAMPLE_NAME.featureCounts.tsv.summary` | featureCounts statistics by sample. |
-| `SAMPLE_NAME.featureCounts.stdout` | featureCounts log file by sample. |
-| `SAMPLE_NAME.featureCounts.tsv` | featureCounts output file by sample.|
-| `Correspondence_global_clstr_contigs.txt` | Correspondance table of inter-sample clusters and initial genes. One line = one correspondance between an inter-sample cluster (first column) and an initial gene (second column). |
-| `Clusters_Count_table_all_samples.txt` | Abundance table of reads. Each cell contains the sum of aligned reads on each initial gene of each inter-sample cluster for each sample (inter_sample clusters in rows and samples in colums).|
+| `SAMPLE_NAME.featureCounts.tsv.summary` | featureCounts statistics by sample. |
+| `SAMPLE_NAME.featureCounts.stdout` | featureCounts log file by sample. |
+| `SAMPLE_NAME.featureCounts.tsv` | featureCounts output file by sample.|
+| `Correspondence_global_clstr_contigs.txt` | Correspondance table of inter-sample clusters and initial genes. One line = one correspondance between an inter-sample cluster (first column) and an initial gene (second column). |
+| `Clusters_Count_table_all_samples.txt` | Abundance table of reads. Each cell contains the sum of aligned reads on each initial gene of each inter-sample cluster for each sample (inter_sample clusters in rows and samples in colums).|
#### **06_func_annot/06_3_functional_annotation/**
| File | Description |
| ----------------------- | --------------------------------------- |
| `SAMPLE_NAME_diamond_one2one.emapper.seed_orthologs` | eggNOG-mapper intermediate file containing seed matches onto eggNOG database. |
-| `SAMPLE_NAME_diamond_one2one.emapper.annotations` | eggNOG-mapper final file containing functional annotations for genes with a matches into eggNOG database. |
-| `SAMPLE_NAME.best_hit` | Diamond best hits results for each gene. Best hits are diamond hits with the maximum bitScore for this gene. |
-| `Quantifications_and_functional_annotations.tsv` | Table where a row corresponds to an inter-sample cluster. Columns corresponds to quantification of the sum of aligned reads on all genes of each inter-sample cluster (columns `*featureCounts.tsv`), sum of abundance in all samples (column `sum`), eggNOG-mapper results (from `seed_eggNOG_ortholog` to `PFAMs` column) and diamond best hits results (last two columns `sseqid` and `stitle` correspond to `diamond_db_id`and `diamond_db_description`). |
-| `GOs_abundance.tsv` | Quantification table storing for each GO term (rows) the sum of aligned reads into all genes having this functional annotation for each sample (columns). |
-| `KEGG_ko_abundance.tsv` | Quantification table storing for each KEGG_ko (rows) the sum of aligned reads into all genes having this functional annotation for each sample (columns). |
-| `KEGG_Pathway_abundance.tsv` | Quantification table storing for each KEGG_Pathway (rows) the sum of aligned reads into all genes having this functional annotation for each sample (columns). |
-| `KEGG_Module_abundance.tsv` | Quantification table storing for each KEGG_Module (rows) the sum of aligned reads into all genes having this functional annotation for each sample (columns). |
-| `PFAM_abundance.tsv` | Quantification table storing for each PFAM (rows) the sum of aligned reads into all genes having this functional annotation for each sample (columns). |
+| `SAMPLE_NAME_diamond_one2one.emapper.annotations` | eggNOG-mapper final file containing functional annotations for genes with a matches into eggNOG database. |
+| `SAMPLE_NAME.best_hit` | Diamond best hits results for each gene. Best hits are diamond hits with the maximum bitScore for this gene. |
+| `Quantifications_and_functional_annotations.tsv` | Table where a row corresponds to an inter-sample cluster. Columns corresponds to quantification of the sum of aligned reads on all genes of each inter-sample cluster (columns `*featureCounts.tsv`), sum of abundance in all samples (column `sum`), eggNOG-mapper results (from `seed_eggNOG_ortholog` to `PFAMs` column) and diamond best hits results (last two columns `sseqid` and `stitle` correspond to `diamond_db_id`and `diamond_db_description`). |
+| `GOs_abundance.tsv` | Quantification table storing for each GO term (rows) the sum of aligned reads into all genes having this functional annotation for each sample (columns). |
+| `KEGG_ko_abundance.tsv` | Quantification table storing for each KEGG_ko (rows) the sum of aligned reads into all genes having this functional annotation for each sample (columns). |
+| `KEGG_Pathway_abundance.tsv` | Quantification table storing for each KEGG_Pathway (rows) the sum of aligned reads into all genes having this functional annotation for each sample (columns). |
+| `KEGG_Module_abundance.tsv` | Quantification table storing for each KEGG_Module (rows) the sum of aligned reads into all genes having this functional annotation for each sample (columns). |
+| `PFAM_abundance.tsv` | Quantification table storing for each PFAM (rows) the sum of aligned reads into all genes having this functional annotation for each sample (columns). |
### **07_taxo_affi/**
@@ -198,21 +199,21 @@ The `results/` directory contains a sub-directory for each step launched:
| File | Description |
| ----------------------- | --------------------------------------- |
-| `SAMPLE_NAME/SAMPLE_NAME.pergene.tsv` | Taxonomic affiliation of genes. One line corresponds to a gene (1st column), its corresponding taxon id (2nd column), its corresponding lineage (3rd column) and the tax ids of each level of this lineage (4th column). |
-| `SAMPLE_NAME/SAMPLE_NAME.warn.tsv` | List of genes with a hit without corresponding taxonomic affiliation. Each line corresponds to a gene (1st column), the reason why the gene is in this list (2nd column) and match ids into the database used during `05_alignment/05_2_database_alignment/` (3rd column). |
-| `SAMPLE_NAME/top_taxons_per_contig.tsv` | Possible affiliation taxons at each rank and for each contig (one line per contig). The score associated with the taxon is indicated in parenthesis. This file can be helpful to investigate contig affiliation. |
-| `SAMPLE_NAME/SAMPLE_NAME.percontig.tsv` | Taxonomic affiliation of contigs. One line corresponds to a contig (1st column), its corresponding taxon id (2nd column), its corresponding lineage (3rd column) and the tax ids of each level of this lineage (4th column). |
-| `SAMPLE_NAME/SAMPLE_NAME_quantif_percontig.tsv` | Quantification table of reads aligned on contigs affiliated to each lineage of the first column. One line = one taxonomic affiliation (1st column, `lineage_by_level`), the corresponding taxon id (2nd column, `consensus_tax_id`), the tax ids of each level of this taxonomic affiliation (3rd column, `tax_id_by_level`), the name of contigs affiliated to this lineage (4th column, `name_contigs`), the number of contigs affiliated to this lineage (5th column, `nb_contigs`), the sum of the number of reads aligned to these contigs (6th column, `nb_reads`) and the mean depth of these contigs (6th column, `depth`). |
-| `SAMPLE_NAME/SAMPLE_NAME_quantif_percontig_by_[taxonomic_level].tsv` | One file by taxonomic level (superkingdom, phylum, order, class, family, genus, species) for the sample `SAMPLE_NAME`. Quantification table of reads aligned on contigs affiliated to each lineage of the corresponding [taxonomic level]. One line = one taxonomic affiliation at this [taxonomic level] with is taxon id (1st column, `tax_id_by_level`), its lineage (2nd column, `lineage_by_level`), the name of contigs affiliated to this lineage (3rd column, `name_contigs`), the number of contigs affiliated to this lineage (4th column, `nb_contigs`), the sum of the number of reads aligned to these contigs (5th column, `nb_reads`) and the mean depth of these contigs (6th column, `depth`). |
+| `SAMPLE_NAME/SAMPLE_NAME.pergene.tsv` | Taxonomic affiliation of genes. One line corresponds to a gene (1st column), its corresponding taxon id (2nd column), its corresponding lineage (3rd column) and the tax ids of each level of this lineage (4th column). |
+| `SAMPLE_NAME/SAMPLE_NAME.warn.tsv` | List of genes with a hit without corresponding taxonomic affiliation. Each line corresponds to a gene (1st column), the reason why the gene is in this list (2nd column) and match ids into the database used during `05_alignment/05_2_database_alignment/` (3rd column). |
+| `SAMPLE_NAME/top_taxons_per_contig.tsv` | Possible affiliation taxons at each rank and for each contig (one line per contig). The score associated with the taxon is indicated in parenthesis. This file can be helpful to investigate contig affiliation. |
+| `SAMPLE_NAME/SAMPLE_NAME.percontig.tsv` | Taxonomic affiliation of contigs. One line corresponds to a contig (1st column), its corresponding taxon id (2nd column), its corresponding lineage (3rd column) and the tax ids of each level of this lineage (4th column). |
+| `SAMPLE_NAME/SAMPLE_NAME_quantif_percontig.tsv` | Quantification table of reads aligned on contigs affiliated to each lineage of the first column. One line = one taxonomic affiliation (1st column, `lineage_by_level`), the corresponding taxon id (2nd column, `consensus_tax_id`), the tax ids of each level of this taxonomic affiliation (3rd column, `tax_id_by_level`), the name of contigs affiliated to this lineage (4th column, `name_contigs`), the number of contigs affiliated to this lineage (5th column, `nb_contigs`), the sum of the number of reads aligned to these contigs (6th column, `nb_reads`) and the mean depth of these contigs (6th column, `depth`). |
+| `SAMPLE_NAME/SAMPLE_NAME_quantif_percontig_by_[taxonomic_level].tsv` | One file by taxonomic level (superkingdom, phylum, order, class, family, genus, species) for the sample `SAMPLE_NAME`. Quantification table of reads aligned on contigs affiliated to each lineage of the corresponding [taxonomic level]. One line = one taxonomic affiliation at this [taxonomic level] with is taxon id (1st column, `tax_id_by_level`), its lineage (2nd column, `lineage_by_level`), the name of contigs affiliated to this lineage (3rd column, `name_contigs`), the number of contigs affiliated to this lineage (4th column, `nb_contigs`), the sum of the number of reads aligned to these contigs (5th column, `nb_reads`) and the mean depth of these contigs (6th column, `depth`). |
| `SAMPLE_NAME/graphs/SAMPLE_NAME_aln_diamond.m8_contig_taxonomy_level.pdf` | Figure representing the number of contigs (y-axis) affiliated to each taxonomy levels (x-axis). |
-| `SAMPLE_NAME/top_taxons_per_contig.tsv` | Details of top possible taxons per contig and for each taxonomic rank. For each rank, there is a list of the top possible taxons and their weigth in parentesis (only taxons with a weigth > 1 is written). For example in the genus column we can have `Escherichia (68.0);Shigella (20.0);Enterobacter (4.0);Salmonella (4.0);Felsduovirus (4.0)`. This file can be useful to understand an affiliation in detail. |
+| `SAMPLE_NAME/top_taxons_per_contig.tsv` | Details of top possible taxons per contig and for each taxonomic rank. For each rank, there is a list of the top possible taxons and their weigth in parentesis (only taxons with a weigth > 1 is written). For example in the genus column we can have `Escherichia (68.0);Shigella (20.0);Enterobacter (4.0);Salmonella (4.0);Felsduovirus (4.0)`. This file can be useful to understand an affiliation in detail. |
#### **07_taxo_affi/07_2_affiliation_merged**
| File | Description |
| ----------------------- | --------------------------------------- |
-| `quantification_by_contig_lineage_all.tsv` | Quantification table of reads aligned on contigs affiliated to each lineage. One line = one taxonomic affiliation with its lineage (1st column, `lineage_by_level`), the taxon id at each level of this lineage (2nd column, `tax_id_by_level`), and then all next 3-columns blocks correspond to one sample. Each 3-column block corresponds to the name of contigs affiliated to this lineage (1st column, `name_contigs_SAMPLE_NAME_quantif_percontig`), the number of contigs affiliated to this lineage (2nd column, `nb_contigs_SAMPLE_NAME_quantif_percontig`), the sum of the number of reads aligned to these contigs (3rd column, `nb_reads_SAMPLE_NAME_quantif_percontig`) and the mean depth of these contigs (4th column, `depth_SAMPLE_NAME_quantif_percontig`). |
+| `quantification_by_contig_lineage_all.tsv` | Quantification table of reads aligned on contigs affiliated to each lineage. One line = one taxonomic affiliation with its lineage (1st column, `lineage_by_level`), the taxon id at each level of this lineage (2nd column, `tax_id_by_level`), and then all next 3-columns blocks correspond to one sample. Each 3-column block corresponds to the name of contigs affiliated to this lineage (1st column, `name_contigs_SAMPLE_NAME_quantif_percontig`), the number of contigs affiliated to this lineage (2nd column, `nb_contigs_SAMPLE_NAME_quantif_percontig`), the sum of the number of reads aligned to these contigs (3rd column, `nb_reads_SAMPLE_NAME_quantif_percontig`) and the mean depth of these contigs (4th column, `depth_SAMPLE_NAME_quantif_percontig`). |
| `quantification_by_contig_lineage_[taxonomic_level].tsv` | One file by taxonomic level (superkingdom, phylum, order, class, family, genus, species). Quantification table of reads aligned on contigs affiliated to each lineage of the corresponding [taxonomic level]. One line = one taxonomic affiliation at this [taxonomic level] with its taxon id (1st column, `tax_id_by_level`), its lineage (2nd column, `lineage_by_level`), and then all next 3-columns blocks correspond to one sample. Each 3-column block corresponds to the name of contigs affiliated to this lineage (1st column, `name_contigs_SAMPLE_NAME_quantif_percontig_by_[taxonomic_level]`), the number of contigs affiliated to this lineage (2nd column, `nb_contigs_SAMPLE_NAME_quantif_percontig_by_[taxonomic_level]`) and the sum of the number of reads aligned to these contigs (3rd column, `nb_reads_SAMPLE_NAME_quantif_percontig_by_[taxonomic_level]`) and the mean depth of these contigs (4th column, `depth_SAMPLE_NAME_quantif_percontig_by_[taxonomic_level]`). |
@@ -222,8 +223,8 @@ The `results/` directory contains a sub-directory for each step launched:
| ----------------------- | --------------------------------------- |
| `krona_mean_depth_abundance.html` | Krona plot of the taxonomic affiliation of contigs. The abundance of a taxon is the percentage of the depth of the contigs affiliated to this taxon. |
| `krona_read_count_abundance.html` | Krona plot of the taxonomic affiliation of contigs. The abundance of a taxon is the percentage of reads mapping the contigs affiliated to this taxon. |
-| `abundance_per_rank.html` | This plot represents the abundance of affiliation made for each taxonomic rank.|
-| `most_abundant_taxa.html` | This plot represents the abundance of the 10 most abundant taxa at each taxonomic rank and for each sample.|
+| `abundance_per_rank.html` | This plot represents the abundance of affiliation made for each taxonomic rank.|
+| `most_abundant_taxa.html` | This plot represents the abundance of the 10 most abundant taxa at each taxonomic rank and for each sample.|
### **08_binning/**
@@ -236,19 +237,19 @@ The `results/` directory contains a sub-directory for each step launched:
| File or directory/ | Description |
| ----------------------- | --------------------------------------- |
-| `SAMPLE_NAME/concoct`| This directory contains bins fasta files from the individual binning tool CONCOCT (see informations [here](https://concoct.readthedocs.io/en/latest/)). |
-| `SAMPLE_NAME/maxbin2`| This directory contains bins fasta files from the individual binning tool MaxBin2(see informations [here](https://academic.oup.com/bioinformatics/article/32/4/605/1744462)). |
-| `SAMPLE_NAME/metabat2`| This directory contains bins fasta files from the individual binning tool MetaBAT2 (see informations [here](https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6662567/)). |
-| `SAMPLE_NAME/bin_refinement`| This directory contains bins fasta files and statistics from the aggregate binning tool BINETTE, which takes into account the bins sets from the 3 individual binning tools to try to improve the quality of the bins (see informations [here](https://forgemia.inra.fr/jean.mainguy/binette)). |
+| `SAMPLE_NAME/concoct` | This directory contains bins fasta files from the individual binning tool CONCOCT (see informations [here](https://concoct.readthedocs.io/en/latest/)). |
+| `SAMPLE_NAME/maxbin2` | This directory contains bins fasta files from the individual binning tool MaxBin2(see informations [here](https://academic.oup.com/bioinformatics/article/32/4/605/1744462)). |
+| `SAMPLE_NAME/metabat2` | This directory contains bins fasta files from the individual binning tool MetaBAT2 (see informations [here](https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6662567/)). |
+| `SAMPLE_NAME/bin_refinement` | This directory contains bins fasta files and statistics from the aggregate binning tool BINETTE, which takes into account the bins sets from the 3 individual binning tools to try to improve the quality of the bins (see informations [here](https://forgemia.inra.fr/jean.mainguy/binette)). |
| `SAMPLE_NAME/bin_refinement/unbinned_contigs.fasta`| Contigs from the sample assembly that are not retrieved in any bin. The proportion of non-binned contigs to the global size of the sample assembly can be seen in the MultiQC report, in the section Bins Size (bp) quality. |
#### **08_binning/08_2_dereplicated_bins**
| File or directory/ | Description |
| ----------------------- | --------------------------------------- |
-| dereplicated_genomes/ | The dRep software allows to compare the bins of all samples in a pair-wise manner, and to clusturize the bins that share similar DNA content in terms of Average Nucleotide Identity (ANI). The threshold of 95% ANI is used to create a set of species-level representative genomes (SRGs). Within each cluster of bins, the best bin in terms of quality (completeness,contamination,N50,strain-heterogeneity) is selected as representative genome. The directory dereplicated_genomes contains all the representative bins that will be used in order to compare abundances of SRGs between the samples. |
-| data_tables/genomeInformation.csv | Calculated metrics of all the bins before de-replication, used to select the representative bin after de-replication. Completeness and contamination where calculated before with CheckM2, during bin_refinement step. dRep adds further metrics and use all four to choose the best bin. |
-| data_tables/Bins_clusters_composition.tsv | Associates each representative bins cluster (1st column) to the bins list within the cluster (2nd column, bins seperates by commas). |
-| figures/ | `Primary_clustering_dendrogram.pdf`: The primary clustering dendrogram summarizes the pair-wise Mash distance between all genomes in the genome list. The first clusturing used Mash, an incredibly fast but not robust algorithm, in order to accelerate the process during the second clusturing. **`Secondary_clustering_dendrograms.pdf`**: Each primary cluster with more than one member will have a page in the Secondary clustering dendrograms file. Bins with more than **95% of ANI distance similarity** (--drep_threshold default value paramater, that correspond to the threshold for separate species.) will be grouped together. **You can try different dRep thresholds to be more or less stringent, for example if the goal of dereplication is to generate a set of genomes that are distinct when mapping short reads, 98% ANI is an appropriate threshold.** You can also try higher threshold in order to separate species that belong to low divergent clades (96, or 97%). `Cluster_scoring.pdf`: Each secondary cluster will have its own page in the Cluster scoring figure. These figures show the score of each genome, as well as all metrics that can go into determin, and will always be the genome with the highest score.`Clustering scatterplots.pdf` provides some information about genome alignment statistics, and `Winning genomes.pdf` provides some information about only the “best†genomes of each replicate set, as well as a couple quick overall statistics.
+| `dereplicated_genomes/` | The dRep software allows to compare the bins of all samples in a pair-wise manner, and to clusturize the bins that share similar DNA content in terms of Average Nucleotide Identity (ANI). The threshold of 95% ANI is used to create a set of species-level representative genomes (SRGs). Within each cluster of bins, the best bin in terms of quality (completeness,contamination,N50,strain-heterogeneity) is selected as representative genome. The directory dereplicated_genomes contains all the representative bins that will be used in order to compare abundances of SRGs between the samples. |
+| `data_tables/genomeInformation.csv` | Calculated metrics of all the bins before de-replication, used to select the representative bin after de-replication. Completeness and contamination where calculated before with CheckM2, during bin_refinement step. dRep adds further metrics and use all four to choose the best bin. |
+| `data_tables/Bins_clusters_composition.tsv` | Associates each representative bins cluster (1st column) to the bins list within the cluster (2nd column, bins seperates by commas). |
+| `figures/ ` | `Primary_clustering_dendrogram.pdf`: The primary clustering dendrogram summarizes the pair-wise Mash distance between all genomes in the genome list. The first clusturing used Mash, an incredibly fast but not robust algorithm, in order to accelerate the process during the second clusturing. **`Secondary_clustering_dendrograms.pdf`**: Each primary cluster with more than one member will have a page in the Secondary clustering dendrograms file. Bins with more than **95% of ANI distance similarity** (--drep_threshold default value paramater, that correspond to the threshold for separate species.) will be grouped together. **You can try different dRep thresholds to be more or less stringent, for example if the goal of dereplication is to generate a set of genomes that are distinct when mapping short reads, 98% ANI is an appropriate threshold.** You can also try higher threshold in order to separate species that belong to low divergent clades (96, or 97%). `Cluster_scoring.pdf`: Each secondary cluster will have its own page in the Cluster scoring figure. These figures show the score of each genome, as well as all metrics that can go into determin, and will always be the genome with the highest score.`Clustering scatterplots.pdf` provides some information about genome alignment statistics, and `Winning genomes.pdf` provides some information about only the “best†genomes of each replicate set, as well as a couple quick overall statistics.
If you want to make further analysis about intra-population genetic diversity (microdiversity) on the genomes, you can use **inStrain** software specifically developed for this purpose. The documentation is here:
@@ -264,27 +265,27 @@ https://instrain.readthedocs.io/en/latest/tutorial.html#quick-start
#### **08_binning/08_3_gtdbtk/**
| File | Description |
| ----------------------- | --------------------------------------- |
-| gtdbtk.bac120.summary.tsv | Taxonomic classifications provided by GTDB-Tk. One line = one bin id (1st column, `user_genome`), its taxonomical classification based on the closest reference genome from the GTDB-Tk database (2nd column, `classification`), the accession number of the closest reference genome (3rd column, `fastani_reference`). Please see GTDB-Tk documentation [here](https://ecogenomics.github.io/GTDBTk/files/summary.tsv.html) for information on additional columns.
+| `gtdbtk.bac120.summary.tsv` | Taxonomic classifications provided by GTDB-Tk. One line = one bin id (1st column, `user_genome`), its taxonomical classification based on the closest reference genome from the GTDB-Tk database (2nd column, `classification`), the accession number of the closest reference genome (3rd column, `fastani_reference`). Please see GTDB-Tk documentation [here](https://ecogenomics.github.io/GTDBTk/files/summary.tsv.html) for information on additional columns.
#### **08_4_mapping_on_final_bins**
| File | Description |
| ----------------------- | --------------------------------------- |
-| mapping/SAMPLE_NAME/ | In order to compare genomes abundances between samples, mapping of metagenomics samples reads against the final set of de-replicated bins is performed.
-| mapping/SAMPLE_NAME/`SAMPLE_NAME.sort.bam` | Alignment of reads on contigs (.bam file). |
-| mapping/SAMPLE_NAME/`SAMPLE_NAME.sort.bam.bai` | Index of .bam file. |
+| `mapping/SAMPLE_NAME/` | In order to compare genomes abundances between samples, mapping of metagenomics samples reads against the final set of de-replicated bins is performed.
+| `mapping/SAMPLE_NAME/SAMPLE_NAME.sort.bam` | Alignment of reads on contigs (.bam file). |
+| `mapping/SAMPLE_NAME/SAMPLE_NAME.sort.bam.bai` | Index of .bam file. |
### **MultiQC/**
| File | Description |
| ----------------------- | --------------------------------------- |
-| `multiqc_report.html` | MultiQC report file containing graphs and a summary of analysis done by metagWGS. |
+| `multiqc_report.html` | MultiQC report file containing graphs and a summary of analysis done by metagWGS. |
### **pipeline_info/**
| File | Description |
| ----------------------- | --------------------------------------- |
| `software_versions.csv` | Indicates the versions of the tools used in the pipeline. |
-| `db_versions.tsv` | Indicates the size, date of last modification and path of the file or folder for each databank used in the pipeline. For the host genome, the number of sequence is in parenthesis with the size of the file. |
+| `db_versions.tsv` | Indicates the size, date of last modification and path of the file or folder for each databank used in the pipeline. For the host genome, the number of sequence is in parenthesis with the size of the file. |
## Description of other files in your working directory (not in `results` directory):
diff --git a/docs/usage.md b/docs/usage.md
index a62d587..6d7bbd6 100644
--- a/docs/usage.md
+++ b/docs/usage.md
@@ -345,7 +345,7 @@ For example check the version of software in the yaml file and the singularity r
* `--gtdbtk_bank`: indicates path to the GTDBTK database.
* `--metabat2_seed`: Set the seed for metabat2, for exact reproducibility of metabat2 (default: 0 (random seed))
-
+* `--min_completeness [nb]`: Minimum % of bins completeness for the bins to be kept after bin_refinement step. Default: 50
* `--binning_cross_alignment ["all","group","individual"]`: defines mapping strategy to compute co-abundances for binning. `all` means that each samples will be mapped against every assembly, `group` means that all sample from a group will be mapped against every assembly of the group, `individual` means that each sample will only be mapped against his assembly. Default `individual`
diff --git a/main.nf b/main.nf
index 51f06c6..1ac4a5c 100644
--- a/main.nf
+++ b/main.nf
@@ -65,7 +65,7 @@ include { MULTIQC } from './modules/multiqc'
--stop_at_assembly Stop the pipeline at this step
--assembly Indicate the assembly tool for short reads ["metaspades" or "megahit" ]. Default: "metaspades".
or for HiFi reads ["hifiasm-meta", "metaflye"]. Default: "hifiasm-meta".
- --coassembly Assemble together samples labeled with the same group in the samplesheet
+ --coassembly Assemble together samples labeled with the same group in the samplesheet.
S03_FILTERING options:
--stop_at_filtering Stop the pipeline at this step
@@ -98,10 +98,7 @@ include { MULTIQC } from './modules/multiqc'
'individual': Each sample is aligned against the assembly of the sample in question
'group': The samples labeled with the same group in the samplesheet will be aligned against each assembly within the group of samples.
'all': All the samples will be aligned against all the assembly (WARNING: It could take a long time.) (default: individual).
- --min_completeness [nb] Minimum % of bins completeness for the bins to be kept after bin_refinement step. Default: 0 (all bins are kept).
- According to MIMAG standards, 'Medium-quality' genomes require to have at least completeness estimates of ≥50% and less than 10% contamination.
- --max_contamination [nb] Maximum % of bins contamination for the bins to be kept after bin_refinement step. Default: 100.
- associated with a maximum contamination of 10% to be considered as "Medium-quality".
+ --min_completeness [nb] Minimum % of bins completeness for the bins to be kept after bin_refinement step. Default: 50.
--drep_threshold [nb] Average Nucleotide Identity (ANI) threshold used for bins de-replication. Default: 0.95 corresponding to 95%. Use a number between 0 and 1.
Most studies agree that 95% ANI is an appropriate threshold for species-level de-replicationton. If the goal of dereplication is to generate a set
of genomes that are distinct when mapping short reads, 98% (0.98) ANI is an appropriate threshold.
diff --git a/nextflow.config b/nextflow.config
index b0f4b71..b5e00d2 100644
--- a/nextflow.config
+++ b/nextflow.config
@@ -7,9 +7,6 @@
params {
// metagWGS parameters.
- reads = ""
- assemblies = ""
- single_end = false
adapter1 = "AGATCGGAAGAGC"
adapter2 = "AGATCGGAAGAGC"
quality_type = "sanger"
--
GitLab
From fa2db3e090a6e7d394b41f0efd63a1566fd78365 Mon Sep 17 00:00:00 2001
From: "maina.vienne" <maina.vienne@inrae.fr>
Date: Mon, 23 Jan 2023 19:40:34 +0100
Subject: [PATCH 188/194] Fix outdated doc
---
README.md | 1 -
1 file changed, 1 deletion(-)
diff --git a/README.md b/README.md
index b15d396..417675f 100644
--- a/README.md
+++ b/README.md
@@ -27,7 +27,6 @@ Many of these steps are optional and their necessity depends on the desired anal
* `S04_STRUCTURAL_ANNOT`
* makes a structural annotation of genes ([Prodigal](https://github.com/hyattpd/Prodigal) + [Barrnap](https://github.com/tseemann/barrnap) + [tRNAscan-SE](https://github.com/UCSC-LoweLab/tRNAscan-SE) + [merge_annotations.py](bin/merge_annotations.py))
* `S05_ALIGNMENT`
- * aligns reads to the contigs ([BWA-MEM2](https://github.com/bwa-mem2/bwa-mem2) or [Minimap2](https://github.com/lh3/minimap2) + [Samtools](http://www.htslib.org/))
* aligns the protein sequence of genes against a protein database ([DIAMOND](https://github.com/bbuchfink/diamond))
* `S06_FUNC_ANNOT`
* makes a sample and global clustering of genes ([cd-hit-est](http://weizhongli-lab.org/cd-hit/) + [cd_hit_produce_table_clstr.py](bin/cd_hit_produce_table_clstr.py))
--
GitLab
From d0b461b1029b9ddcba57394bced09cfd215fb095 Mon Sep 17 00:00:00 2001
From: "maina.vienne" <maina.vienne@inrae.fr>
Date: Tue, 24 Jan 2023 14:51:03 +0100
Subject: [PATCH 189/194] Fix coassembly for hifi and quast
---
main.nf | 7 ++++---
subworkflows/02_assembly.nf | 2 +-
2 files changed, 5 insertions(+), 4 deletions(-)
diff --git a/main.nf b/main.nf
index 1ac4a5c..4b02999 100644
--- a/main.nf
+++ b/main.nf
@@ -372,6 +372,7 @@ workflow {
ch_unfilter_assembly_flagstat_report = S02_ASSEMBLY.out.flagstat
ch_quast_before_filter_report = S02_ASSEMBLY.out.assembly_report
+ ch_quast = ch_quast_before_filter_report
ch_software_versions_S02 = S02_ASSEMBLY.out.software_versions
ch_circular = S02_ASSEMBLY.out.circular
@@ -398,6 +399,7 @@ workflow {
ch_quast_after_filter_report = S03_FILTERING.out.quast_report
+ ch_quast = ch_quast_after_filter_report
ch_sam_coverage = S03_FILTERING.out.sam_coverage
@@ -445,8 +447,7 @@ workflow {
}
if ( !params.stop_at_clean && !params.stop_at_assembly && !params.stop_at_filtering && !params.stop_at_structural_annot && !params.skip_binning ) {
- ch_quast=ch_quast_after_filter_report.ifEmpty(ch_quast_before_filter_report)
-
+
S08_BINNING( ch_reads, ch_assembly, ch_bam, ch_gtbdtk_db, ch_quast, ch_circular)
ch_bins_abundances_report = S08_BINNING.out.bins_abundances_report
ch_bins_stats_report = S08_BINNING.out.bins_stats_report
@@ -460,7 +461,7 @@ workflow {
ch_software_versions_S07,
ch_software_versions_S08).unique { it.getBaseName() }.collect()
- GET_SOFTWARE_VERSIONS(ch_software_versions_total) // ch_v_eggnogmapper.ifEmpty([]).first()
+ GET_SOFTWARE_VERSIONS(ch_software_versions_total)
ch_software_versions = GET_SOFTWARE_VERSIONS.out.yaml
MULTIQC (
diff --git a/subworkflows/02_assembly.nf b/subworkflows/02_assembly.nf
index 94e1771..fa446b7 100644
--- a/subworkflows/02_assembly.nf
+++ b/subworkflows/02_assembly.nf
@@ -73,7 +73,7 @@ workflow STEP_02_ASSEMBLY {
if (params.type.toUpperCase() == "SR") {
return [meta, fastq.collect { it[0] }, fastq.collect { it[1] }]
} else {
- return [meta, fastq ]
+ return [meta, fastq.flatten().sort{ it.baseName }]
}}
.set { ch_reads_assembly }
--
GitLab
From aaeb617a79e440dea4c5199dd6edabc851080f99 Mon Sep 17 00:00:00 2001
From: "maina.vienne" <maina.vienne@inrae.fr>
Date: Tue, 24 Jan 2023 15:28:48 +0100
Subject: [PATCH 190/194] Update docs
---
README.md | 5 +++--
docs/README.md | 2 +-
docs/output.md | 12 ++++++------
docs/usage.md | 7 ++++---
4 files changed, 14 insertions(+), 12 deletions(-)
diff --git a/README.md b/README.md
index 417675f..442feb2 100644
--- a/README.md
+++ b/README.md
@@ -21,7 +21,8 @@ Many of these steps are optional and their necessity depends on the desired anal
* `S02_ASSEMBLY`
* assembles reads ([metaSPAdes](https://github.com/ablab/spades) or [Megahit](https://github.com/voutcn/megahit) or [Hifiasm_meta](https://github.com/lh3/hifiasm-meta) or [metaFlye](https://github.com/fenderglass/Flye))
* assesses the quality of assembly ([metaQUAST](http://quast.sourceforge.net/metaquast))
- * reads deduplication ([BWA-MEM2](https://github.com/bwa-mem2/bwa-mem2) or [Minimap2](https://github.com/lh3/minimap2) + [Samtools](http://www.htslib.org/))
+ * reads deduplication, alignment against contigs for Short reads([BWA-MEM2](https://github.com/bwa-mem2/bwa-mem2) + [Samtools](http://www.htslib.org/))
+ * reads alignment against contigs for HiFi reads ([Minimap2](https://github.com/lh3/minimap2) + [Samtools](http://www.htslib.org/))
* `S03_FILTERING`
* filters contigs with low CPM value ([Filter_contig_per_cpm.py](bin/Filter_contig_per_cpm.py) + [metaQUAST](http://quast.sourceforge.net/metaquast))
* `S04_STRUCTURAL_ANNOT`
@@ -51,7 +52,7 @@ A report html file is generated at the end of the workflow with [MultiQC](https:
The pipeline is built using [Nextflow,](https://www.nextflow.io/docs/latest/index.html#) a bioinformatics workflow tool to run tasks across multiple compute infrastructures in a very portable manner.
-Three [Singularity](https://sylabs.io/docs/) containers are available making installation trivial and results highly reproducible.
+Two [Singularity](https://sylabs.io/docs/) containers are available making installation trivial and results highly reproducible.
## Documentation
diff --git a/docs/README.md b/docs/README.md
index c93cda9..8e75987 100644
--- a/docs/README.md
+++ b/docs/README.md
@@ -9,7 +9,7 @@ The metagWGS documentation can be found in the following pages:
* An overview of how the pipeline works, how to run it and a description of all of the different command-line flags.
* [Output](output.md)
* An overview of the different output files and directories produced by the pipeline.
- * [Use case](use_case.md)
+ * [Use case](use_case.md) (WARNING: not up-to-date, needs to be updated)
* A tutorial to learn how to launch the pipeline on a test dataset on [genologin cluster](http://bioinfo.genotoul.fr/).
* [Functional tests](../functional_tests/README.md)
* (for developers) A tool to launch a new version of the pipeline on curated input data and compare its results with known output.
diff --git a/docs/output.md b/docs/output.md
index f32070e..ef6ddaa 100644
--- a/docs/output.md
+++ b/docs/output.md
@@ -59,7 +59,7 @@ The `results/` directory contains a sub-directory for each step launched:
| File or directory/ | Description |
| ----------------------- | --------------------------------------- |
| `cleaned_SAMPLE_NAME_R{1,2}.fastq.gz` | There are one R1 and one R2 file for each sample. |
-| `logs/` | Contains cutadapt (`SAMPLE_NAME_cutadapt.log`) and sickle (`SAMPLE_NAME_sickle.log`) log files for each sample. Only if you remove host reads, in `SAMPLE_NAME_cleaned_R{1,2}.nb_bases` you have the number of nucleotides into each cleaned R1 and R2 files of each sample. Only if you remove host reads, you also have a samtools flagstat file for each sample before removing host reads (`SAMPLE_NAME.no_filter.flagstat`) and into the directory `host_filter_flagstat/` there are the samtools flagstat files (`SAMPLE_NAME.host_filter.flagstat`) after removing host reads. |
+| `logs/` | Contains cutadapt (`SAMPLE_NAME_cutadapt.log`) and sickle (`SAMPLE_NAME_sickle.log`) log files for each sample. Only if you remove host reads, in `SAMPLE_NAME_cleaned_R{1,2}.nb_bases` you have the number of nucleotides into each cleaned R1 and R2 files of each sample. Only if you remove host reads, you also have a samtools flagstat file for each sample before removing host reads (`SAMPLE_NAME.no_filter.flagstat`) and into the directory `host_filter_flagstat/` there are the samtools flagstat files (`SAMPLE_NAME.host_filter.flagstat`) after removing host reads. |
#### **01_clean_qc/01_2_qc/**
@@ -85,11 +85,11 @@ The `results/` directory contains a sub-directory for each step launched:
| File or directory/ | Description |
|--------------------------------|----------------------------------------------------------------------------------------------------------------------------------|
-| `SAMPLE_NAME.fna` | unfiltered assembly (primary assembly) with renamed contigs. Contig names follow this pattern: `<sample_name>_c<contig_number>`. |
-| `assembly_metric` | Contains metaQUAST quality control files of contigs. |
-| `<assembly_tool>_SAMPLE_NAME/` | output files of the assembly tool. It contains logs and original fasta files of the assembly before renaming. |
+| `SAMPLE_NAME.fna` | unfiltered assembly (primary assembly) with renamed contigs. Contig names follow this pattern: `<sample_name>_c<contig_number>`. |
+| `assembly_metric` | Contains metaQUAST quality control files of contigs. |
+| `<assembly_tool>_SAMPLE_NAME/` | output files of the assembly tool. It contains logs and original fasta files of the assembly before renaming. |
| `SAMPLE_NAME_original_to_new_contig_name.tsv` | Tabular file with two fields: original contig name from the assembly tool and the new contig name given by the pipeline. |
-| `circular_contigs/` | only for HiFi:
+| `circular_contigs/` | only for HiFi: contains the fasta file of each circular contigs, useful for bin refinement in the binning step |
#### **02_assembly/02_2_deduplicated_reads**
@@ -246,7 +246,7 @@ The `results/` directory contains a sub-directory for each step launched:
#### **08_binning/08_2_dereplicated_bins**
| File or directory/ | Description |
| ----------------------- | --------------------------------------- |
-| `dereplicated_genomes/` | The dRep software allows to compare the bins of all samples in a pair-wise manner, and to clusturize the bins that share similar DNA content in terms of Average Nucleotide Identity (ANI). The threshold of 95% ANI is used to create a set of species-level representative genomes (SRGs). Within each cluster of bins, the best bin in terms of quality (completeness,contamination,N50,strain-heterogeneity) is selected as representative genome. The directory dereplicated_genomes contains all the representative bins that will be used in order to compare abundances of SRGs between the samples. |
+| `dereplicated_genomes/` | The dRep software allows to compare the bins of all samples in a pair-wise manner, and to clusterize the bins that share similar DNA content in terms of Average Nucleotide Identity (ANI). The threshold of 95% ANI is used to create a set of species-level representative genomes (SRGs). Within each cluster of bins, the best bin in terms of quality (completeness,contamination,N50,strain-heterogeneity) is selected as representative genome. The directory dereplicated_genomes contains all the representative bins that will be used in order to compare abundances of SRGs between the samples. |
| `data_tables/genomeInformation.csv` | Calculated metrics of all the bins before de-replication, used to select the representative bin after de-replication. Completeness and contamination where calculated before with CheckM2, during bin_refinement step. dRep adds further metrics and use all four to choose the best bin. |
| `data_tables/Bins_clusters_composition.tsv` | Associates each representative bins cluster (1st column) to the bins list within the cluster (2nd column, bins seperates by commas). |
| `figures/ ` | `Primary_clustering_dendrogram.pdf`: The primary clustering dendrogram summarizes the pair-wise Mash distance between all genomes in the genome list. The first clusturing used Mash, an incredibly fast but not robust algorithm, in order to accelerate the process during the second clusturing. **`Secondary_clustering_dendrograms.pdf`**: Each primary cluster with more than one member will have a page in the Secondary clustering dendrograms file. Bins with more than **95% of ANI distance similarity** (--drep_threshold default value paramater, that correspond to the threshold for separate species.) will be grouped together. **You can try different dRep thresholds to be more or less stringent, for example if the goal of dereplication is to generate a set of genomes that are distinct when mapping short reads, 98% ANI is an appropriate threshold.** You can also try higher threshold in order to separate species that belong to low divergent clades (96, or 97%). `Cluster_scoring.pdf`: Each secondary cluster will have its own page in the Cluster scoring figure. These figures show the score of each genome, as well as all metrics that can go into determin, and will always be the genome with the highest score.`Clustering scatterplots.pdf` provides some information about genome alignment statistics, and `Winning genomes.pdf` provides some information about only the “best†genomes of each replicate set, as well as a couple quick overall statistics.
diff --git a/docs/usage.md b/docs/usage.md
index 6d7bbd6..4979893 100644
--- a/docs/usage.md
+++ b/docs/usage.md
@@ -197,7 +197,7 @@ You can use a `stop_at_[STEP]` parameter to launch only the steps leading to and
`--stop_at_[STEP]`: indicate the step of the pipeline you want to stop at. The steps available are described in the [`README`](../README.md#metagwgs-steps) (`S01_CLEAN_QC`, `S02_ASSEMBLY`, `S03_FILTERING`, `S04_STRUCTURAL_ANNOT`).
-**NOTE: `S05_ALIGNMENT`, `S06_FUNC_ANNOT` and `S07_TAXO_AFFI` being the 3 last steps, there is no `--stop_at_[STEP]`; see 'Skip' subsection for more information.**
+**NOTE: `S05_ALIGNMENT`, `S06_FUNC_ANNOT`, `S07_TAXO_AFFI` and `S08_BINNING` being the 4 last steps, there is no `--stop_at_[STEP]`; see 'Skip' subsection for more information.**
For each [STEP](../README.md#metagwgs-steps), specific parameters are available. You can add them to the command line and run the pipeline. They are described in the section [other parameters step by step](usage.md#other-parameters-step-by-step).
@@ -279,7 +279,7 @@ No parameter available for this substep.
* `--coassembly` allows to assemble together the samples labeled with the same group in the samplesheet. It will generate one assembly for each group. To co-assemble all of your samples together, you must indicate a unique group for each sample in the samplesheet. **WARNING** With the coassembly, you can't use `--binning_cross_alignment 'group'` because one binning will be generate for each group co-assembled and automatically mapping with every sample of his group but `--binning_cross_alignment 'all'` can be use to cross align every sample with every group.
-**WARNING 4:** For short reads, the user can choose between `metaspades` or `megahit` for `--assembly` parameter. The choice can be based on CPUs and memory availability: `metaspades` needs more CPUs and memory than `megahit` but our tests showed that assembly metrics are better for `metaspades` than `megahit`.For PacBio HiFi reads, the user can choose between `hifiasm-meta` or `metaflye`.
+**WARNING 4:** For short reads, the user can choose between `metaspades` or `megahit` for `--assembly` parameter. The choice can be based on CPUs and memory availability: `metaspades` needs more CPUs and memory than `megahit` but our tests showed that assembly metrics are better for `metaspades` than `megahit`. For PacBio HiFi reads, the user can choose between `hifiasm-meta` or `metaflye`.
**Note:** you may need to tweak the memory and cpus settings of the Nextflow process, especially if you are using `metaspades`. If this is the case, create a `nextflow.config` file in our working directory and modify these parameters (be aware that the memory must be in GB) such as :
```bash
@@ -348,6 +348,7 @@ For example check the version of software in the yaml file and the singularity r
* `--min_completeness [nb]`: Minimum % of bins completeness for the bins to be kept after bin_refinement step. Default: 50
* `--binning_cross_alignment ["all","group","individual"]`: defines mapping strategy to compute co-abundances for binning. `all` means that each samples will be mapped against every assembly, `group` means that all sample from a group will be mapped against every assembly of the group, `individual` means that each sample will only be mapped against his assembly. Default `individual`
+**WARNING 13:** CheckM2 has reproducibility issues, therefore this step is not perfectly reproducible from one run to another. Results may vary slightly.
#### Others parameters
@@ -373,4 +374,4 @@ See the description of output files in [this part](output.md) of the documentati
> If you have an account on the [genologin cluster](http://bioinfo.genotoul.fr/) and you would like to familiarize yourself with metagWGS, see the tutorial available in the [use case documentation page](use_case.md). It allows the analysis of big test datasets with metagWGS. (WARNING: use_case documentation is not up-to-date, needs to be updated)
-**WARNING 13:** the test dataset in `metagwgs-test-datasets/small` used in [I. Basic Usage](usage.md#i-basic-usage) is a small test dataset which allows to test all steps but with few CPUs and memory.
+**WARNING 14:** the test dataset in `metagwgs-test-datasets/small` used in [I. Basic Usage](usage.md#i-basic-usage) is a small test dataset which allows to test all steps but with few CPUs and memory.
--
GitLab
From 107d1e2e664445988aa431b8473cd84301815343 Mon Sep 17 00:00:00 2001
From: "maina.vienne" <maina.vienne@inrae.fr>
Date: Tue, 24 Jan 2023 16:45:59 +0100
Subject: [PATCH 191/194] Add metro map + update doc, version, config test
---
README.md | 7 ++++---
conf/functional_test.config | 4 ++--
docs/metagwgs_metro_map.png | Bin 0 -> 328464 bytes
functional_tests/README.md | 8 +++-----
nextflow.config | 2 +-
5 files changed, 10 insertions(+), 11 deletions(-)
create mode 100644 docs/metagwgs_metro_map.png
diff --git a/README.md b/README.md
index 442feb2..5aaa0f5 100644
--- a/README.md
+++ b/README.md
@@ -6,7 +6,8 @@
### Pipeline graphical representation
The workflow processes raw data from `.fastq/.fastq.gz` input and/or assemblies (contigs) `.fa/.fasta` and uses the modules represented in this figure:
-
+
+
### metagWGS steps
@@ -21,13 +22,13 @@ Many of these steps are optional and their necessity depends on the desired anal
* `S02_ASSEMBLY`
* assembles reads ([metaSPAdes](https://github.com/ablab/spades) or [Megahit](https://github.com/voutcn/megahit) or [Hifiasm_meta](https://github.com/lh3/hifiasm-meta) or [metaFlye](https://github.com/fenderglass/Flye))
* assesses the quality of assembly ([metaQUAST](http://quast.sourceforge.net/metaquast))
- * reads deduplication, alignment against contigs for Short reads([BWA-MEM2](https://github.com/bwa-mem2/bwa-mem2) + [Samtools](http://www.htslib.org/))
+ * reads deduplication, alignment against contigs for short reads ([BWA-MEM2](https://github.com/bwa-mem2/bwa-mem2) + [Samtools](http://www.htslib.org/))
* reads alignment against contigs for HiFi reads ([Minimap2](https://github.com/lh3/minimap2) + [Samtools](http://www.htslib.org/))
* `S03_FILTERING`
* filters contigs with low CPM value ([Filter_contig_per_cpm.py](bin/Filter_contig_per_cpm.py) + [metaQUAST](http://quast.sourceforge.net/metaquast))
* `S04_STRUCTURAL_ANNOT`
* makes a structural annotation of genes ([Prodigal](https://github.com/hyattpd/Prodigal) + [Barrnap](https://github.com/tseemann/barrnap) + [tRNAscan-SE](https://github.com/UCSC-LoweLab/tRNAscan-SE) + [merge_annotations.py](bin/merge_annotations.py))
-* `S05_ALIGNMENT`
+* `S05_PROTEIN_ALIGNMENT`
* aligns the protein sequence of genes against a protein database ([DIAMOND](https://github.com/bbuchfink/diamond))
* `S06_FUNC_ANNOT`
* makes a sample and global clustering of genes ([cd-hit-est](http://weizhongli-lab.org/cd-hit/) + [cd_hit_produce_table_clstr.py](bin/cd_hit_produce_table_clstr.py))
diff --git a/conf/functional_test.config b/conf/functional_test.config
index 6268237..fd02222 100644
--- a/conf/functional_test.config
+++ b/conf/functional_test.config
@@ -5,9 +5,9 @@ singularity.enabled = true
singularity.autoMounts = true
process {
- container = "/work/project/plateforme/metaG/functional_test/singularity_img/dev_v2.4/metagwgs.sif"
+ container = "/work/project/plateforme/metaG/functional_test/singularity_img/v2.4/metagwgs.sif"
withLabel: BINNING {
- container = "/work/project/plateforme/metaG/functional_test/singularity_img/binning.sif"
+ container = "/work/project/plateforme/metaG/functional_test/singularity_img/v2.4/binning.sif"
}
}
diff --git a/docs/metagwgs_metro_map.png b/docs/metagwgs_metro_map.png
new file mode 100644
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diff --git a/functional_tests/README.md b/functional_tests/README.md
index 561c00e..8e2fa42 100644
--- a/functional_tests/README.md
+++ b/functional_tests/README.md
@@ -22,7 +22,7 @@ sbatch $METAG_PATH/functional_tests/launch_example.sh
```bash
module load system/Python-3.7.4
-python $METAG_PATH/functional_tests/main.py -step 07_taxo_affi -exp_dir /work/project/plateforme/metaG/functional_test/metagwgs-test-datasets/small/output -obs_dir ./results
+python $METAG_PATH/functional_tests/main.py -step 08_binning -exp_dir /work/project/plateforme/metaG/functional_test/metagwgs-test-datasets/small/output -obs_dir ./results
```
Don't forget to modify the `METAG_PATH` variable, if metagwgs is not in your working directory.
@@ -33,7 +33,7 @@ If you do not have access to genologin or the directory, you need to download th
### Donwload of DB and data
1. Install metagwgs as described here: [installation doc](../docs/installation.md)
-2. Get datasets: two datasets are currently available for these functional tests at https://forgemia.inra.fr/genotoul-bioinfo/metagwgs-test-datasets.git
+2. Get datasets: three datasets are currently available for these functional tests at https://forgemia.inra.fr/genotoul-bioinfo/metagwgs-test-datasets.git
```bash
git clone git@forgemia.inra.fr:genotoul-bioinfo/metagwgs-test-datasets.git
@@ -91,9 +91,7 @@ python $METAG_PATH/functional_tests/main.py -step 07_taxo_affi -exp_dir $DATASET
This procedure allows you to perform functional tests for small data (Short reads), to test HiFi data you need to include the parameters `--input "/work/project/plateforme/metaG/functional_test/metagwgs-test-datasets/hifi/input/samplesheet.csv" --type "HiFi" ` to the launch_example.sh script. And change the expected directory for the python command.
-## III. Test skips and check processes
-
-Besoin d'un cluster absolument
+## III. Test skips and check processes (WARNING: not up-to-date, needs to be updated, will be replaced by dry-run)
The script `test_parameters_and_processes.py` check if execution with parameters specified in `expected_processes.tsv`, run processes as expected.
diff --git a/nextflow.config b/nextflow.config
index b5e00d2..2d6f062 100644
--- a/nextflow.config
+++ b/nextflow.config
@@ -98,5 +98,5 @@ manifest {
description = 'Analysis of metagenomics data'
mainScript = 'main.nf'
nextflowVersion = '>=20.01.0'
- version = '2.3.1'
+ version = '2.4'
}
--
GitLab
From d9c4db465be173f08d954491303d20b143d26cbc Mon Sep 17 00:00:00 2001
From: Jean Mainguy <jean.mainguy@inra.fr>
Date: Tue, 24 Jan 2023 19:47:08 +0100
Subject: [PATCH 192/194] Update README.md
---
README.md | 2 +-
1 file changed, 1 insertion(+), 1 deletion(-)
diff --git a/README.md b/README.md
index 5aaa0f5..b69d6eb 100644
--- a/README.md
+++ b/README.md
@@ -42,7 +42,7 @@ Many of these steps are optional and their necessity depends on the desired anal
* aligns reads samples against assemblies (according to the strategy used) ([BWA-MEM2](https://github.com/bwa-mem2/bwa-mem2) or [Minimap2](https://github.com/lh3/minimap2))
* performs metagenome binning ([METABAT2](https://bitbucket.org/berkeleylab/metabat/src/master/) + [MAXBIN2](https://sourceforge.net/projects/maxbin/) + [CONCOCT](https://github.com/BinPro/CONCOCT))
- * refines bin sets ([BINETTE](https://forgemia.inra.fr/jean.mainguy/binette/))
+ * refines bin sets ([BINETTE](https://github.com/genotoul-bioinfo/Binette))
* dereplicates bins between samples ([DREP](https://github.com/MrOlm/drep))
* taxonomically affiliates the bins ([GTDBTK](https://github.com/Ecogenomics/GTDBTk))
* calculates bins abundances between samples ([BWA-MEM2](https://github.com/bwa-mem2/bwa-mem2) or [Minimap2](https://github.com/lh3/minimap2) + [SAMTOOLS](http://www.htslib.org/))
--
GitLab
From 5cf6ce2ed439c108d9072c3ec8c7a8dda8088a42 Mon Sep 17 00:00:00 2001
From: Maina Vienne <maina.vienne@inrae.fr>
Date: Thu, 26 Jan 2023 14:40:01 +0100
Subject: [PATCH 193/194] Update docs Add missing parameters on docs
---
docs/output.md | 2 +-
docs/usage.md | 4 ++++
main.nf | 1 +
3 files changed, 6 insertions(+), 1 deletion(-)
diff --git a/docs/output.md b/docs/output.md
index ef6ddaa..c7e6aad 100644
--- a/docs/output.md
+++ b/docs/output.md
@@ -240,7 +240,7 @@ The `results/` directory contains a sub-directory for each step launched:
| `SAMPLE_NAME/concoct` | This directory contains bins fasta files from the individual binning tool CONCOCT (see informations [here](https://concoct.readthedocs.io/en/latest/)). |
| `SAMPLE_NAME/maxbin2` | This directory contains bins fasta files from the individual binning tool MaxBin2(see informations [here](https://academic.oup.com/bioinformatics/article/32/4/605/1744462)). |
| `SAMPLE_NAME/metabat2` | This directory contains bins fasta files from the individual binning tool MetaBAT2 (see informations [here](https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6662567/)). |
-| `SAMPLE_NAME/bin_refinement` | This directory contains bins fasta files and statistics from the aggregate binning tool BINETTE, which takes into account the bins sets from the 3 individual binning tools to try to improve the quality of the bins (see informations [here](https://forgemia.inra.fr/jean.mainguy/binette)). |
+| `SAMPLE_NAME/bin_refinement` | This directory contains bins fasta files and statistics from the aggregate binning tool BINETTE, which takes into account the bins sets from the 3 individual binning tools to try to improve the quality of the bins (see informations [here](https://github.com/genotoul-bioinfo/Binette)). |
| `SAMPLE_NAME/bin_refinement/unbinned_contigs.fasta`| Contigs from the sample assembly that are not retrieved in any bin. The proportion of non-binned contigs to the global size of the sample assembly can be seen in the MultiQC report, in the section Bins Size (bp) quality. |
#### **08_binning/08_2_dereplicated_bins**
diff --git a/docs/usage.md b/docs/usage.md
index 4979893..41cb601 100644
--- a/docs/usage.md
+++ b/docs/usage.md
@@ -345,8 +345,12 @@ For example check the version of software in the yaml file and the singularity r
* `--gtdbtk_bank`: indicates path to the GTDBTK database.
* `--metabat2_seed`: Set the seed for metabat2, for exact reproducibility of metabat2 (default: 0 (random seed))
+
* `--min_completeness [nb]`: Minimum % of bins completeness for the bins to be kept after bin_refinement step. Default: 50
+
* `--binning_cross_alignment ["all","group","individual"]`: defines mapping strategy to compute co-abundances for binning. `all` means that each samples will be mapped against every assembly, `group` means that all sample from a group will be mapped against every assembly of the group, `individual` means that each sample will only be mapped against his assembly. Default `individual`
+
+* `--drep_threshold [nb]`: Average Nucleotide Identity (ANI) threshold used for bins de-replication. Default: 0.95 corresponding to 95%. Use a number between 0 and 1. Most studies agree that 95% ANI is an appropriate threshold for species-level de-replicationton. If the goal of dereplication is to generate a set of genomes that are distinct when mapping short reads, 98% (0.98) ANI is an appropriate threshold.
**WARNING 13:** CheckM2 has reproducibility issues, therefore this step is not perfectly reproducible from one run to another. Results may vary slightly.
diff --git a/main.nf b/main.nf
index 4b02999..d2c0456 100644
--- a/main.nf
+++ b/main.nf
@@ -94,6 +94,7 @@ include { MULTIQC } from './modules/multiqc'
SO8_BINNING options:
--gtdbtk_bank Path to the GTDBTK database
--skip_binning Skip this step
+ --metabat2_seed Set the seed for metabat2, for exact reproducibility of metabat2 (default: 0 (random seed))
--binning_cross_alignment Mapping strategy to compute co-abundances for binning: .
'individual': Each sample is aligned against the assembly of the sample in question
'group': The samples labeled with the same group in the samplesheet will be aligned against each assembly within the group of samples.
--
GitLab
From 2fd73f919ec1d97d3d63186ff4ef2038a787b9ed Mon Sep 17 00:00:00 2001
From: Maina Vienne <maina.vienne@inrae.fr>
Date: Thu, 26 Jan 2023 16:12:00 +0100
Subject: [PATCH 194/194] Replace bin refinement by BINETTE on binning.png
---
docs/08_binning.png | Bin 138817 -> 223297 bytes
docs/binning.svg | 2177 +++++++++++++++++++++++++++++++++++++++++++
2 files changed, 2177 insertions(+)
create mode 100644 docs/binning.svg
diff --git a/docs/08_binning.png b/docs/08_binning.png
index cc99305629db199489061ec612bd30d9a694c8eb..e5b2addb722833a9a6ea9c54b2c28c6049f51abe 100644
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diff --git a/docs/binning.svg b/docs/binning.svg
new file mode 100644
index 0000000..0681d7a
--- /dev/null
+++ b/docs/binning.svg
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+ xmlns:inkscape="http://www.inkscape.org/namespaces/inkscape"
+ version="1.1"
+ viewBox="0 0 1200 408"
+ fill="none"
+ stroke="none"
+ stroke-linecap="square"
+ stroke-miterlimit="10"
+ id="svg673"
+ sodipodi:docname="binning.svg"
+ inkscape:version="1.0.2-2 (e86c870879, 2021-01-15)"
+ width="12.5in"
+ height="4.25in">
+ <metadata
+ id="metadata679">
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+ rdf:about="">
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+ <dc:type
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+ style="font-size:9.33333px;text-align:center;text-anchor:middle;fill:#fff9ff;fill-opacity:1">Sample 1</tspan><tspan
+ sodipodi:role="line"
+ x="86.289764"
+ y="102.21074"
+ style="font-size:9.33333px;text-align:center;text-anchor:middle;fill:#fff9ff;fill-opacity:1"
+ id="tspan3854">reads</tspan><tspan
+ sodipodi:role="line"
+ x="87.586311"
+ y="113.87741"
+ style="font-size:9.33333px;fill:#fff9ff;fill-opacity:1"
+ id="tspan3856"><tspan
+ style="text-align:center;text-anchor:middle;fill:#fff9ff;fill-opacity:1"
+ id="tspan3858">(.fq)</tspan> </tspan></text>
+ <path
+ style="fill:#25a632;fill-opacity:0.596078;fill-rule:evenodd;stroke:none"
+ clip-path="url(#clipEmfPath1-8-6-3)"
+ d="m -0.07999584,11.439406 c 0,-6.3596698 5.15973204,-11.51940184 11.51940184,-11.51940184 h 46.077607 c 6.369669,0 11.519401,5.15973204 11.519401,11.51940184 v 94.235104 c 0,6.36967 -5.149732,11.5194 -11.519401,11.5194 H 11.439406 c -6.3596698,0 -11.51940184,-5.14973 -11.51940184,-11.5194 z"
+ id="path1751-9-0-1"
+ transform="matrix(0.73172884,0,0,0.32087103,61.183931,250.14713)" />
+ <text
+ xml:space="preserve"
+ style="font-style:normal;font-variant:normal;font-weight:700;font-size:9.33333px;line-height:125%;font-family:Arial;text-align:start;letter-spacing:0px;word-spacing:0px;text-anchor:start;fill:#fff9ff;fill-opacity:1;stroke:none"
+ x="87.084045"
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+ id="text1757-0-3-5"><tspan
+ sodipodi:role="line"
+ x="87.084045"
+ y="260.98883"
+ id="tspan1755-8-4-9"
+ style="font-size:9.33333px;text-align:center;text-anchor:middle;fill:#fff9ff;fill-opacity:1">Sample 1</tspan><tspan
+ sodipodi:role="line"
+ x="87.084045"
+ y="272.65549"
+ style="font-size:9.33333px;text-align:center;text-anchor:middle;fill:#fff9ff;fill-opacity:1"
+ id="tspan3854-9">assembly</tspan><tspan
+ sodipodi:role="line"
+ x="88.380592"
+ y="284.32214"
+ style="font-size:9.33333px;fill:#fff9ff;fill-opacity:1"
+ id="tspan3856-2"><tspan
+ style="text-align:center;text-anchor:middle;fill:#fff9ff;fill-opacity:1"
+ id="tspan3858-6">(.fna)</tspan> </tspan></text>
+ <path
+ style="fill:#25a632;fill-opacity:0.596078;fill-rule:evenodd;stroke:none"
+ clip-path="url(#clipEmfPath1-8-6-3-3)"
+ d="m -0.07999584,11.439406 c 0,-6.3596698 5.15973204,-11.51940184 11.51940184,-11.51940184 h 46.077607 c 6.369669,0 11.519401,5.15973204 11.519401,11.51940184 v 94.235104 c 0,6.36967 -5.149732,11.5194 -11.519401,11.5194 H 11.439406 c -6.3596698,0 -11.51940184,-5.14973 -11.51940184,-11.5194 z"
+ id="path1751-9-0-1-8"
+ transform="matrix(0.73172884,0,0,0.32087103,111.75841,250.14713)" />
+ <text
+ xml:space="preserve"
+ style="font-style:normal;font-variant:normal;font-weight:700;font-size:9.33333px;line-height:125%;font-family:Arial;text-align:start;letter-spacing:0px;word-spacing:0px;text-anchor:start;fill:#fff9ff;fill-opacity:1;stroke:none"
+ x="137.65852"
+ y="260.98883"
+ id="text1757-0-3-5-0"><tspan
+ sodipodi:role="line"
+ x="137.65852"
+ y="260.98883"
+ id="tspan1755-8-4-9-5"
+ style="font-size:9.33333px;text-align:center;text-anchor:middle;fill:#fff9ff;fill-opacity:1">Sample 2</tspan><tspan
+ sodipodi:role="line"
+ x="137.65852"
+ y="272.65549"
+ style="font-size:9.33333px;text-align:center;text-anchor:middle;fill:#fff9ff;fill-opacity:1"
+ id="tspan3854-9-2">assembly</tspan><tspan
+ sodipodi:role="line"
+ x="137.65852"
+ y="284.32214"
+ style="font-size:9.33333px;fill:#fff9ff;fill-opacity:1"
+ id="tspan3856-2-7" /></text>
+ <path
+ style="fill:#25a632;fill-opacity:0.596078;fill-rule:evenodd;stroke:none"
+ clip-path="url(#clipEmfPath1-8-6-3-3-6)"
+ d="m -0.07999584,11.439406 c 0,-6.3596698 5.15973204,-11.51940184 11.51940184,-11.51940184 h 46.077607 c 6.369669,0 11.519401,5.15973204 11.519401,11.51940184 v 94.235104 c 0,6.36967 -5.149732,11.5194 -11.519401,11.5194 H 11.439406 c -6.3596698,0 -11.51940184,-5.14973 -11.51940184,-11.5194 z"
+ id="path1751-9-0-1-8-2"
+ transform="matrix(0.73172884,0,0,0.32087103,162.33288,250.14713)" />
+ <text
+ xml:space="preserve"
+ style="font-style:normal;font-variant:normal;font-weight:700;font-size:9.33333px;line-height:125%;font-family:Arial;text-align:start;letter-spacing:0px;word-spacing:0px;text-anchor:start;fill:#fff9ff;fill-opacity:1;stroke:none"
+ x="188.233"
+ y="260.98883"
+ id="text1757-0-3-5-0-6"><tspan
+ sodipodi:role="line"
+ x="188.233"
+ y="260.98883"
+ id="tspan1755-8-4-9-5-8"
+ style="font-size:9.33333px;text-align:center;text-anchor:middle;fill:#fff9ff;fill-opacity:1">Sample 3</tspan><tspan
+ sodipodi:role="line"
+ x="188.233"
+ y="272.65549"
+ style="font-size:9.33333px;text-align:center;text-anchor:middle;fill:#fff9ff;fill-opacity:1"
+ id="tspan3854-9-2-6">assembly</tspan><tspan
+ sodipodi:role="line"
+ x="188.233"
+ y="284.32214"
+ style="font-size:9.33333px;fill:#fff9ff;fill-opacity:1"
+ id="tspan3856-2-7-9" /></text>
+ <path
+ style="fill:#f9d1ba;fill-opacity:1;fill-rule:evenodd;stroke:none"
+ clip-path="url(#clipEmfPath1-8-6-3-9)"
+ d="m -0.07999584,11.439406 c 0,-6.3596698 5.15973204,-11.51940184 11.51940184,-11.51940184 h 46.077607 c 6.369669,0 11.519401,5.15973204 11.519401,11.51940184 v 94.235104 c 0,6.36967 -5.149732,11.5194 -11.519401,11.5194 H 11.439406 c -6.3596698,0 -11.51940184,-5.14973 -11.51940184,-11.5194 z"
+ id="path1751-9-0-1-1"
+ transform="matrix(0.73172884,0,0,0.32087103,257.9902,247.50134)" />
+ <text
+ xml:space="preserve"
+ style="font-style:normal;font-variant:normal;font-weight:700;font-size:9.33333px;line-height:125%;font-family:Arial;text-align:start;letter-spacing:0px;word-spacing:0px;text-anchor:start;fill:#fff9ff;fill-opacity:1;stroke:none"
+ x="283.89032"
+ y="258.34302"
+ id="text1757-0-3-5-2"><tspan
+ sodipodi:role="line"
+ x="283.89032"
+ y="258.34302"
+ id="tspan1755-8-4-9-6"
+ style="font-size:9.33333px;text-align:center;text-anchor:middle;fill:#fff9ff;fill-opacity:1">Sample 1</tspan><tspan
+ sodipodi:role="line"
+ x="283.89032"
+ y="270.00967"
+ style="font-size:9.33333px;text-align:center;text-anchor:middle;fill:#fff9ff;fill-opacity:1"
+ id="tspan3854-9-7">assembly</tspan><tspan
+ sodipodi:role="line"
+ x="283.89032"
+ y="281.67633"
+ style="font-size:9.33333px;fill:#fff9ff;fill-opacity:1"
+ id="tspan3856-2-9" /></text>
+ <path
+ style="fill:#f9d1ba;fill-opacity:1;fill-rule:evenodd;stroke:none"
+ clip-path="url(#clipEmfPath1-8-6-3-3-65)"
+ d="m -0.07999584,11.439406 c 0,-6.3596698 5.15973204,-11.51940184 11.51940184,-11.51940184 h 46.077607 c 6.369669,0 11.519401,5.15973204 11.519401,11.51940184 v 94.235104 c 0,6.36967 -5.149732,11.5194 -11.519401,11.5194 H 11.439406 c -6.3596698,0 -11.51940184,-5.14973 -11.51940184,-11.5194 z"
+ id="path1751-9-0-1-8-9"
+ transform="matrix(0.73172884,0,0,0.32087103,308.56467,247.50134)" />
+ <text
+ xml:space="preserve"
+ style="font-style:normal;font-variant:normal;font-weight:700;font-size:9.33333px;line-height:125%;font-family:Arial;text-align:start;letter-spacing:0px;word-spacing:0px;text-anchor:start;fill:#fff9ff;fill-opacity:1;stroke:none"
+ x="334.46478"
+ y="258.34302"
+ id="text1757-0-3-5-0-8"><tspan
+ sodipodi:role="line"
+ x="334.46478"
+ y="258.34302"
+ id="tspan1755-8-4-9-5-7"
+ style="font-size:9.33333px;text-align:center;text-anchor:middle;fill:#fff9ff;fill-opacity:1">Sample 2</tspan><tspan
+ sodipodi:role="line"
+ x="334.46478"
+ y="270.00967"
+ style="font-size:9.33333px;text-align:center;text-anchor:middle;fill:#fff9ff;fill-opacity:1"
+ id="tspan3854-9-2-9">assembly</tspan><tspan
+ sodipodi:role="line"
+ x="334.46478"
+ y="281.67633"
+ style="font-size:9.33333px;fill:#fff9ff;fill-opacity:1"
+ id="tspan3856-2-7-3" /></text>
+ <path
+ style="fill:#b8cff1;fill-opacity:1;fill-rule:evenodd;stroke:none"
+ clip-path="url(#clipEmfPath1-8-6-3-3-6-5)"
+ d="m -0.07999584,11.439406 c 0,-6.3596698 5.15973204,-11.51940184 11.51940184,-11.51940184 h 46.077607 c 6.369669,0 11.519401,5.15973204 11.519401,11.51940184 v 94.235104 c 0,6.36967 -5.149732,11.5194 -11.519401,11.5194 H 11.439406 c -6.3596698,0 -11.51940184,-5.14973 -11.51940184,-11.5194 z"
+ id="path1751-9-0-1-8-2-3"
+ transform="matrix(0.73172884,0,0,0.32087103,359.13914,247.50134)" />
+ <text
+ xml:space="preserve"
+ style="font-style:normal;font-variant:normal;font-weight:700;font-size:9.33333px;line-height:125%;font-family:Arial;text-align:start;letter-spacing:0px;word-spacing:0px;text-anchor:start;fill:#fff9ff;fill-opacity:1;stroke:none"
+ x="385.03928"
+ y="258.34302"
+ id="text1757-0-3-5-0-6-5"><tspan
+ sodipodi:role="line"
+ x="385.03928"
+ y="258.34302"
+ id="tspan1755-8-4-9-5-8-3"
+ style="font-size:9.33333px;text-align:center;text-anchor:middle;fill:#fff9ff;fill-opacity:1">Sample 3</tspan><tspan
+ sodipodi:role="line"
+ x="385.03928"
+ y="270.00967"
+ style="font-size:9.33333px;text-align:center;text-anchor:middle;fill:#fff9ff;fill-opacity:1"
+ id="tspan3854-9-2-6-1">assembly</tspan><tspan
+ sodipodi:role="line"
+ x="385.03928"
+ y="281.67633"
+ style="font-size:9.33333px;fill:#fff9ff;fill-opacity:1"
+ id="tspan3856-2-7-9-8" /></text>
+ <path
+ style="fill:#56d1b4;fill-opacity:1;fill-rule:evenodd;stroke:none"
+ clip-path="url(#clipEmfPath1-8-6-3-1)"
+ d="m -0.07999584,11.439406 c 0,-6.3596698 5.15973204,-11.51940184 11.51940184,-11.51940184 h 46.077607 c 6.369669,0 11.519401,5.15973204 11.519401,11.51940184 v 94.235104 c 0,6.36967 -5.149732,11.5194 -11.519401,11.5194 H 11.439406 c -6.3596698,0 -11.51940184,-5.14973 -11.51940184,-11.5194 z"
+ id="path1751-9-0-1-5"
+ transform="matrix(0.73172884,0,0,0.32087103,455.76248,247.50135)" />
+ <text
+ xml:space="preserve"
+ style="font-style:normal;font-variant:normal;font-weight:700;font-size:9.33333px;line-height:125%;font-family:Arial;text-align:start;letter-spacing:0px;word-spacing:0px;text-anchor:start;fill:#fff9ff;fill-opacity:1;stroke:none"
+ x="481.6626"
+ y="258.34302"
+ id="text1757-0-3-5-9"><tspan
+ sodipodi:role="line"
+ x="481.6626"
+ y="258.34302"
+ id="tspan1755-8-4-9-8"
+ style="font-size:9.33333px;text-align:center;text-anchor:middle;fill:#fff9ff;fill-opacity:1">Sample 1</tspan><tspan
+ sodipodi:role="line"
+ x="481.6626"
+ y="270.00967"
+ style="font-size:9.33333px;text-align:center;text-anchor:middle;fill:#fff9ff;fill-opacity:1"
+ id="tspan3854-9-1">assembly</tspan><tspan
+ sodipodi:role="line"
+ x="481.6626"
+ y="281.67633"
+ style="font-size:9.33333px;fill:#fff9ff;fill-opacity:1"
+ id="tspan3856-2-5" /></text>
+ <path
+ style="fill:#009999;fill-opacity:1;fill-rule:evenodd;stroke:none"
+ clip-path="url(#clipEmfPath1-8-6-3-3-0)"
+ d="m -0.07999584,11.439406 c 0,-6.3596698 5.15973204,-11.51940184 11.51940184,-11.51940184 h 46.077607 c 6.369669,0 11.519401,5.15973204 11.519401,11.51940184 v 94.235104 c 0,6.36967 -5.149732,11.5194 -11.519401,11.5194 H 11.439406 c -6.3596698,0 -11.51940184,-5.14973 -11.51940184,-11.5194 z"
+ id="path1751-9-0-1-8-6"
+ transform="matrix(0.73172884,0,0,0.32087103,506.33696,247.50135)" />
+ <text
+ xml:space="preserve"
+ style="font-style:normal;font-variant:normal;font-weight:700;font-size:9.33333px;line-height:125%;font-family:Arial;text-align:start;letter-spacing:0px;word-spacing:0px;text-anchor:start;fill:#fff9ff;fill-opacity:1;stroke:none"
+ x="532.23706"
+ y="258.34302"
+ id="text1757-0-3-5-0-5"><tspan
+ sodipodi:role="line"
+ x="532.23706"
+ y="258.34302"
+ id="tspan1755-8-4-9-5-6"
+ style="font-size:9.33333px;text-align:center;text-anchor:middle;fill:#fff9ff;fill-opacity:1">Sample 2</tspan><tspan
+ sodipodi:role="line"
+ x="532.23706"
+ y="270.00967"
+ style="font-size:9.33333px;text-align:center;text-anchor:middle;fill:#fff9ff;fill-opacity:1"
+ id="tspan3854-9-2-64">assembly</tspan><tspan
+ sodipodi:role="line"
+ x="532.23706"
+ y="281.67633"
+ style="font-size:9.33333px;fill:#fff9ff;fill-opacity:1"
+ id="tspan3856-2-7-5" /></text>
+ <path
+ style="fill:#3c88b4;fill-opacity:1;fill-rule:evenodd;stroke:none"
+ clip-path="url(#clipEmfPath1-8-6-3-3-6-6)"
+ d="m -0.07999584,11.439406 c 0,-6.3596698 5.15973204,-11.51940184 11.51940184,-11.51940184 h 46.077607 c 6.369669,0 11.519401,5.15973204 11.519401,11.51940184 v 94.235104 c 0,6.36967 -5.149732,11.5194 -11.519401,11.5194 H 11.439406 c -6.3596698,0 -11.51940184,-5.14973 -11.51940184,-11.5194 z"
+ id="path1751-9-0-1-8-2-5"
+ transform="matrix(0.73172884,0,0,0.32087103,556.91143,247.50135)" />
+ <text
+ xml:space="preserve"
+ style="font-style:normal;font-variant:normal;font-weight:700;font-size:9.33333px;line-height:125%;font-family:Arial;text-align:start;letter-spacing:0px;word-spacing:0px;text-anchor:start;fill:#fff9ff;fill-opacity:1;stroke:none"
+ x="582.81152"
+ y="258.34302"
+ id="text1757-0-3-5-0-6-7"><tspan
+ sodipodi:role="line"
+ x="582.81152"
+ y="258.34302"
+ id="tspan1755-8-4-9-5-8-8"
+ style="font-size:9.33333px;text-align:center;text-anchor:middle;fill:#fff9ff;fill-opacity:1">Sample 3</tspan><tspan
+ sodipodi:role="line"
+ x="582.81152"
+ y="270.00967"
+ style="font-size:9.33333px;text-align:center;text-anchor:middle;fill:#fff9ff;fill-opacity:1"
+ id="tspan3854-9-2-6-0">assembly</tspan><tspan
+ sodipodi:role="line"
+ x="582.81152"
+ y="281.67633"
+ style="font-size:9.33333px;fill:#fff9ff;fill-opacity:1"
+ id="tspan3856-2-7-9-1" /></text>
+ <path
+ style="fill:#25a632;fill-opacity:0.596078;fill-rule:evenodd;stroke:none"
+ clip-path="url(#clipEmfPath1-8-6-35)"
+ d="m -0.07999584,11.439406 c 0,-6.3596698 5.15973204,-11.51940184 11.51940184,-11.51940184 h 46.077607 c 6.369669,0 11.519401,5.15973204 11.519401,11.51940184 v 94.235104 c 0,6.36967 -5.149732,11.5194 -11.519401,11.5194 H 11.439406 c -6.3596698,0 -11.51940184,-5.14973 -11.51940184,-11.5194 z"
+ id="path1751-9-0-4"
+ transform="matrix(0.73172884,0,0,0.32087103,110.96412,79.7023)" />
+ <text
+ xml:space="preserve"
+ style="font-style:normal;font-variant:normal;font-weight:700;font-size:9.33333px;line-height:125%;font-family:Arial;text-align:start;letter-spacing:0px;word-spacing:0px;text-anchor:start;fill:#fff9ff;fill-opacity:1;stroke:none"
+ x="136.86424"
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+ id="text1757-0-3-55"><tspan
+ sodipodi:role="line"
+ x="136.86424"
+ y="90.544083"
+ id="tspan1755-8-4-2"
+ style="font-size:9.33333px;text-align:center;text-anchor:middle;fill:#fff9ff;fill-opacity:1">Sample 2</tspan><tspan
+ sodipodi:role="line"
+ x="136.86424"
+ y="102.21074"
+ style="font-size:9.33333px;text-align:center;text-anchor:middle;fill:#fff9ff;fill-opacity:1"
+ id="tspan3854-3">reads</tspan><tspan
+ sodipodi:role="line"
+ x="136.86424"
+ y="113.87741"
+ style="font-size:9.33333px;fill:#fff9ff;fill-opacity:1"
+ id="tspan3856-7" /></text>
+ <path
+ style="fill:#25a632;fill-opacity:0.596078;fill-rule:evenodd;stroke:none"
+ clip-path="url(#clipEmfPath1-8-6-35-5)"
+ d="m -0.07999584,11.439406 c 0,-6.3596698 5.15973204,-11.51940184 11.51940184,-11.51940184 h 46.077607 c 6.369669,0 11.519401,5.15973204 11.519401,11.51940184 v 94.235104 c 0,6.36967 -5.149732,11.5194 -11.519401,11.5194 H 11.439406 c -6.3596698,0 -11.51940184,-5.14973 -11.51940184,-11.5194 z"
+ id="path1751-9-0-4-9"
+ transform="matrix(0.73172884,0,0,0.32087103,161.53859,79.7023)" />
+ <text
+ xml:space="preserve"
+ style="font-style:normal;font-variant:normal;font-weight:700;font-size:9.33333px;line-height:125%;font-family:Arial;text-align:start;letter-spacing:0px;word-spacing:0px;text-anchor:start;fill:#fff9ff;fill-opacity:1;stroke:none"
+ x="187.43872"
+ y="90.544083"
+ id="text1757-0-3-55-0"><tspan
+ sodipodi:role="line"
+ x="187.43872"
+ y="90.544083"
+ id="tspan1755-8-4-2-6"
+ style="font-size:9.33333px;text-align:center;text-anchor:middle;fill:#fff9ff;fill-opacity:1">Sample 3</tspan><tspan
+ sodipodi:role="line"
+ x="187.43872"
+ y="102.21074"
+ style="font-size:9.33333px;text-align:center;text-anchor:middle;fill:#fff9ff;fill-opacity:1"
+ id="tspan3854-3-8">reads</tspan><tspan
+ sodipodi:role="line"
+ x="187.43872"
+ y="113.87741"
+ style="font-size:9.33333px;fill:#fff9ff;fill-opacity:1"
+ id="tspan3856-7-0" /></text>
+ <path
+ style="fill:#f9d1ba;fill-opacity:1;fill-rule:evenodd;stroke:none"
+ clip-path="url(#clipEmfPath1-8-6-2)"
+ d="m -0.07999584,11.439406 c 0,-6.3596698 5.15973204,-11.51940184 11.51940184,-11.51940184 h 46.077607 c 6.369669,0 11.519401,5.15973204 11.519401,11.51940184 v 94.235104 c 0,6.36967 -5.149732,11.5194 -11.519401,11.5194 H 11.439406 c -6.3596698,0 -11.51940184,-5.14973 -11.51940184,-11.5194 z"
+ id="path1751-9-0-8"
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+ <text
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+ style="font-style:normal;font-variant:normal;font-weight:700;font-size:9.33333px;line-height:125%;font-family:Arial;text-align:start;letter-spacing:0px;word-spacing:0px;text-anchor:start;fill:#fff9ff;fill-opacity:1;stroke:none"
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+ y="89.175735"
+ id="tspan1755-8-4-3"
+ style="font-size:9.33333px;text-align:center;text-anchor:middle;fill:#fff9ff;fill-opacity:1">Sample 1</tspan><tspan
+ sodipodi:role="line"
+ x="284.08115"
+ y="100.84239"
+ style="font-size:9.33333px;text-align:center;text-anchor:middle;fill:#fff9ff;fill-opacity:1"
+ id="tspan3854-36">reads</tspan><tspan
+ sodipodi:role="line"
+ x="284.08115"
+ y="112.50906"
+ style="font-size:9.33333px;fill:#fff9ff;fill-opacity:1"
+ id="tspan3856-1" /></text>
+ <path
+ style="fill:#f9d1ba;fill-opacity:1;fill-rule:evenodd;stroke:none"
+ clip-path="url(#clipEmfPath1-8-6-35-8)"
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+ id="path1751-9-0-4-92"
+ transform="matrix(0.73172884,0,0,0.32087103,308.75546,78.33391)" />
+ <text
+ xml:space="preserve"
+ style="font-style:normal;font-variant:normal;font-weight:700;font-size:9.33333px;line-height:125%;font-family:Arial;text-align:start;letter-spacing:0px;word-spacing:0px;text-anchor:start;fill:#fff9ff;fill-opacity:1;stroke:none"
+ x="334.65561"
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+ x="334.65561"
+ y="89.175735"
+ id="tspan1755-8-4-2-62"
+ style="font-size:9.33333px;text-align:center;text-anchor:middle;fill:#fff9ff;fill-opacity:1">Sample 2</tspan><tspan
+ sodipodi:role="line"
+ x="334.65561"
+ y="100.84239"
+ style="font-size:9.33333px;text-align:center;text-anchor:middle;fill:#fff9ff;fill-opacity:1"
+ id="tspan3854-3-83">reads</tspan><tspan
+ sodipodi:role="line"
+ x="334.65561"
+ y="112.50906"
+ style="font-size:9.33333px;fill:#fff9ff;fill-opacity:1"
+ id="tspan3856-7-4" /></text>
+ <path
+ style="fill:#b8cff1;fill-opacity:1;fill-rule:evenodd;stroke:none"
+ clip-path="url(#clipEmfPath1-8-6-35-5-0)"
+ d="m -0.07999584,11.439406 c 0,-6.3596698 5.15973204,-11.51940184 11.51940184,-11.51940184 h 46.077607 c 6.369669,0 11.519401,5.15973204 11.519401,11.51940184 v 94.235104 c 0,6.36967 -5.149732,11.5194 -11.519401,11.5194 H 11.439406 c -6.3596698,0 -11.51940184,-5.14973 -11.51940184,-11.5194 z"
+ id="path1751-9-0-4-9-8"
+ transform="matrix(0.73172884,0,0,0.32087103,359.32993,78.33391)" />
+ <text
+ xml:space="preserve"
+ style="font-style:normal;font-variant:normal;font-weight:700;font-size:9.33333px;line-height:125%;font-family:Arial;text-align:start;letter-spacing:0px;word-spacing:0px;text-anchor:start;fill:#fff9ff;fill-opacity:1;stroke:none"
+ x="385.2301"
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+ id="text1757-0-3-55-0-2"><tspan
+ sodipodi:role="line"
+ x="385.2301"
+ y="89.175735"
+ id="tspan1755-8-4-2-6-2"
+ style="font-size:9.33333px;text-align:center;text-anchor:middle;fill:#fff9ff;fill-opacity:1">Sample 3</tspan><tspan
+ sodipodi:role="line"
+ x="385.2301"
+ y="100.84239"
+ style="font-size:9.33333px;text-align:center;text-anchor:middle;fill:#fff9ff;fill-opacity:1"
+ id="tspan3854-3-8-1">reads</tspan><tspan
+ sodipodi:role="line"
+ x="385.2301"
+ y="112.50906"
+ style="font-size:9.33333px;fill:#fff9ff;fill-opacity:1"
+ id="tspan3856-7-0-7" /></text>
+ <path
+ style="fill:#56d1b4;fill-opacity:1;fill-rule:evenodd;stroke:none"
+ clip-path="url(#clipEmfPath1-8-6-2-9)"
+ d="m -0.07999584,11.439406 c 0,-6.3596698 5.15973204,-11.51940184 11.51940184,-11.51940184 h 46.077607 c 6.369669,0 11.519401,5.15973204 11.519401,11.51940184 v 94.235104 c 0,6.36967 -5.149732,11.5194 -11.519401,11.5194 H 11.439406 c -6.3596698,0 -11.51940184,-5.14973 -11.51940184,-11.5194 z"
+ id="path1751-9-0-8-8"
+ transform="matrix(0.73172884,0,0,0.32087103,453.68494,80.20476)" />
+ <text
+ xml:space="preserve"
+ style="font-style:normal;font-variant:normal;font-weight:700;font-size:9.33333px;line-height:125%;font-family:Arial;text-align:start;letter-spacing:0px;word-spacing:0px;text-anchor:start;fill:#fff9ff;fill-opacity:1;stroke:none"
+ x="479.58508"
+ y="91.046585"
+ id="text1757-0-3-9-7"><tspan
+ sodipodi:role="line"
+ x="479.58508"
+ y="91.046585"
+ id="tspan1755-8-4-3-4"
+ style="font-size:9.33333px;text-align:center;text-anchor:middle;fill:#fff9ff;fill-opacity:1">Sample 1</tspan><tspan
+ sodipodi:role="line"
+ x="479.58508"
+ y="102.71324"
+ style="font-size:9.33333px;text-align:center;text-anchor:middle;fill:#fff9ff;fill-opacity:1"
+ id="tspan3854-36-3">reads</tspan><tspan
+ sodipodi:role="line"
+ x="479.58508"
+ y="114.37991"
+ style="font-size:9.33333px;fill:#fff9ff;fill-opacity:1"
+ id="tspan3856-1-8" /></text>
+ <path
+ style="fill:#009999;fill-opacity:1;fill-rule:evenodd;stroke:none"
+ clip-path="url(#clipEmfPath1-8-6-35-8-9)"
+ d="m -0.07999584,11.439406 c 0,-6.3596698 5.15973204,-11.51940184 11.51940184,-11.51940184 h 46.077607 c 6.369669,0 11.519401,5.15973204 11.519401,11.51940184 v 94.235104 c 0,6.36967 -5.149732,11.5194 -11.519401,11.5194 H 11.439406 c -6.3596698,0 -11.51940184,-5.14973 -11.51940184,-11.5194 z"
+ id="path1751-9-0-4-92-5"
+ transform="matrix(0.73172884,0,0,0.32087103,504.25941,80.20476)" />
+ <text
+ xml:space="preserve"
+ style="font-style:normal;font-variant:normal;font-weight:700;font-size:9.33333px;line-height:125%;font-family:Arial;text-align:start;letter-spacing:0px;word-spacing:0px;text-anchor:start;fill:#fff9ff;fill-opacity:1;stroke:none"
+ x="530.15955"
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+ id="text1757-0-3-55-1-7"><tspan
+ sodipodi:role="line"
+ x="530.15955"
+ y="91.046585"
+ id="tspan1755-8-4-2-62-7"
+ style="font-size:9.33333px;text-align:center;text-anchor:middle;fill:#fff9ff;fill-opacity:1">Sample 2</tspan><tspan
+ sodipodi:role="line"
+ x="530.15955"
+ y="102.71324"
+ style="font-size:9.33333px;text-align:center;text-anchor:middle;fill:#fff9ff;fill-opacity:1"
+ id="tspan3854-3-83-3">reads</tspan><tspan
+ sodipodi:role="line"
+ x="530.15955"
+ y="114.37991"
+ style="font-size:9.33333px;fill:#fff9ff;fill-opacity:1"
+ id="tspan3856-7-4-6" /></text>
+ <path
+ style="fill:#3c88b4;fill-opacity:1;fill-rule:evenodd;stroke:none"
+ clip-path="url(#clipEmfPath1-8-6-35-5-0-7)"
+ d="m -0.07999584,11.439406 c 0,-6.3596698 5.15973204,-11.51940184 11.51940184,-11.51940184 h 46.077607 c 6.369669,0 11.519401,5.15973204 11.519401,11.51940184 v 94.235104 c 0,6.36967 -5.149732,11.5194 -11.519401,11.5194 H 11.439406 c -6.3596698,0 -11.51940184,-5.14973 -11.51940184,-11.5194 z"
+ id="path1751-9-0-4-9-8-3"
+ transform="matrix(0.73172884,0,0,0.32087103,554.83388,80.20476)" />
+ <text
+ xml:space="preserve"
+ style="font-style:normal;font-variant:normal;font-weight:700;font-size:9.33333px;line-height:125%;font-family:Arial;text-align:start;letter-spacing:0px;word-spacing:0px;text-anchor:start;fill:#fff9ff;fill-opacity:1;stroke:none"
+ x="580.73401"
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+ id="text1757-0-3-55-0-2-8"><tspan
+ sodipodi:role="line"
+ x="580.73401"
+ y="91.046585"
+ id="tspan1755-8-4-2-6-2-9"
+ style="font-size:9.33333px;text-align:center;text-anchor:middle;fill:#fff9ff;fill-opacity:1">Sample 3</tspan><tspan
+ sodipodi:role="line"
+ x="580.73401"
+ y="102.71324"
+ style="font-size:9.33333px;text-align:center;text-anchor:middle;fill:#fff9ff;fill-opacity:1"
+ id="tspan3854-3-8-1-4">reads</tspan><tspan
+ sodipodi:role="line"
+ x="580.73401"
+ y="114.37991"
+ style="font-size:9.33333px;fill:#fff9ff;fill-opacity:1"
+ id="tspan3856-7-0-7-1" /></text>
+ <path
+ style="fill:#7dc884;fill-opacity:1;fill-rule:evenodd;stroke:none"
+ clip-path="url(#clipEmfPath1-8-6-3-1-0)"
+ d="m -0.07999584,11.439406 c 0,-6.3596698 5.15973204,-11.51940184 11.51940184,-11.51940184 h 46.077607 c 6.369669,0 11.519401,5.15973204 11.519401,11.51940184 v 94.235104 c 0,6.36967 -5.149732,11.5194 -11.519401,11.5194 H 11.439406 c -6.3596698,0 -11.51940184,-5.14973 -11.51940184,-11.5194 z"
+ id="path1751-9-0-1-5-6"
+ transform="matrix(0.73172884,0,0,0.32087103,1014.9403,71.78593)" />
+ <text
+ xml:space="preserve"
+ style="font-style:normal;font-variant:normal;font-weight:700;font-size:9.33333px;line-height:125%;font-family:Arial;text-align:start;letter-spacing:0px;word-spacing:0px;text-anchor:start;fill:#fff9ff;fill-opacity:1;stroke:none"
+ x="1040.8405"
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+ id="text1757-0-3-5-9-1"><tspan
+ sodipodi:role="line"
+ x="1040.8405"
+ y="82.62764"
+ id="tspan1755-8-4-9-8-5"
+ style="font-size:9.33333px;text-align:center;text-anchor:middle;fill:#fff9ff;fill-opacity:1">Sample 1</tspan><tspan
+ sodipodi:role="line"
+ x="1040.8405"
+ y="94.294296"
+ style="font-size:9.33333px;text-align:center;text-anchor:middle;fill:#fff9ff;fill-opacity:1"
+ id="tspan3854-9-1-6">alignment</tspan><tspan
+ sodipodi:role="line"
+ x="1040.8405"
+ y="105.96097"
+ style="font-size:9.33333px;text-align:center;text-anchor:middle;fill:#fff9ff;fill-opacity:1"
+ id="tspan4696">(.bam)</tspan><tspan
+ sodipodi:role="line"
+ x="1040.8405"
+ y="117.62762"
+ style="font-size:9.33333px;fill:#fff9ff;fill-opacity:1"
+ id="tspan3856-2-5-0" /></text>
+ <path
+ style="fill:#7dc884;fill-opacity:1;fill-rule:evenodd;stroke:none"
+ clip-path="url(#clipEmfPath1-8-6-3-3-0-3)"
+ d="m -0.07999584,11.439406 c 0,-6.3596698 5.15973204,-11.51940184 11.51940184,-11.51940184 h 46.077607 c 6.369669,0 11.519401,5.15973204 11.519401,11.51940184 v 94.235104 c 0,6.36967 -5.149732,11.5194 -11.519401,11.5194 H 11.439406 c -6.3596698,0 -11.51940184,-5.14973 -11.51940184,-11.5194 z"
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+ <text
+ xml:space="preserve"
+ style="font-style:normal;font-variant:normal;font-weight:700;font-size:9.33333px;line-height:125%;font-family:Arial;text-align:start;letter-spacing:0px;word-spacing:0px;text-anchor:start;fill:#fff9ff;fill-opacity:1;stroke:none"
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+ id="text1757-0-3-5-0-5-8"><tspan
+ sodipodi:role="line"
+ x="1091.4149"
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+ id="tspan1755-8-4-9-5-6-8"
+ style="font-size:9.33333px;text-align:center;text-anchor:middle;fill:#fff9ff;fill-opacity:1">Sample 2</tspan><tspan
+ sodipodi:role="line"
+ x="1091.4149"
+ y="94.294296"
+ style="font-size:9.33333px;text-align:center;text-anchor:middle;fill:#fff9ff;fill-opacity:1"
+ id="tspan3854-9-2-64-1">alignment</tspan><tspan
+ sodipodi:role="line"
+ x="1091.4149"
+ y="105.96097"
+ style="font-size:9.33333px;fill:#fff9ff;fill-opacity:1"
+ id="tspan3856-2-7-5-3" /></text>
+ <path
+ style="fill:#7dc884;fill-opacity:1;fill-rule:evenodd;stroke:none"
+ clip-path="url(#clipEmfPath1-8-6-3-3-6-6-0)"
+ d="m -0.07999584,11.439406 c 0,-6.3596698 5.15973204,-11.51940184 11.51940184,-11.51940184 h 46.077607 c 6.369669,0 11.519401,5.15973204 11.519401,11.51940184 v 94.235104 c 0,6.36967 -5.149732,11.5194 -11.519401,11.5194 H 11.439406 c -6.3596698,0 -11.51940184,-5.14973 -11.51940184,-11.5194 z"
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+ <text
+ xml:space="preserve"
+ style="font-style:normal;font-variant:normal;font-weight:700;font-size:9.33333px;line-height:125%;font-family:Arial;text-align:start;letter-spacing:0px;word-spacing:0px;text-anchor:start;fill:#fff9ff;fill-opacity:1;stroke:none"
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+ sodipodi:role="line"
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+ id="tspan1755-8-4-9-5-8-8-0"
+ style="font-size:9.33333px;text-align:center;text-anchor:middle;fill:#fff9ff;fill-opacity:1">Sample 3</tspan><tspan
+ sodipodi:role="line"
+ x="1141.9894"
+ y="94.294296"
+ style="font-size:9.33333px;text-align:center;text-anchor:middle;fill:#fff9ff;fill-opacity:1"
+ id="tspan3854-9-2-6-0-7">alignment</tspan><tspan
+ sodipodi:role="line"
+ x="1141.9894"
+ y="105.96097"
+ style="font-size:9.33333px;fill:#fff9ff;fill-opacity:1"
+ id="tspan3856-2-7-9-1-0" /></text>
+ </g>
+</svg>
--
GitLab