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main.nf
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#!/usr/bin/env nextflow
nextflow.enable.dsl=2
/*
========================================================================================
MET analysis pipeline
========================================================================================
#### Documentation
#### Authors
Valentine Murigneux <[email protected]>
Dean Basic <[email protected]>
========================================================================================
*/
def helpMessage() {
log.info"""
=========================================
MET analysis pipeline v${workflow.manifest.version}
=========================================
Usage:
nextflow main.nf --fqdir /path/to/fastq/directory/ --outdir /path/to/outdir/
Required arguments:
--fqdir Path to the directory containing the PacBio subreads bam files
--outdir Path to the output directory to be created
Optional parameters:
--threads Number of threads (default=16)
Porechop:
--porechop_args Porechop optional parameters (default=""), see https://github.com/rrwick/Porechop#full-usage
--porechop_threads Number of threads for Porechop (default=4)
--skip_porechop Skip the Porechop trimming step (default=false)
Adaptive Read Sequencing:
--skip_adaptive_sampling_metrics Skip the Adaptive sampling metrics step (default=false)
--nanocomp_threads Number of threads for NanoComp (default=4)
Mapping:
--minimap_threads Number of threads for Minimap2 (default=12)
Flye Assembly:
--flye_args Flye optional parameters (default=`--flye_args "-meta"), see [details](https://github.com/fenderglass/Flye/blob/flye/docs/USAGE.md)
--flye_threads Number of threads for Flye (default=?)
--memory Memory usage for Flye (default=0)
--skip_assembly Skip the metagenome assembly step (default=false)
Centrifuge taxonomy classification:
--skip_download_centrifuge_db Skip the centrifuge database downloading step (default=false)
--skip_centrifuge Skip the centrifuge taxonomy classification step (default=false)
--centrifuge_db Path to download the centrifuge database (default='https://genome-idx.s3.amazonaws.com/centrifuge/nt_2018_3_3.tar.gz')
--centrifuge_reference_tax_ID Taxonomy ID for reference ID (default="")
--skip_centrifuge_remove_contaminated Skip the removal of centrifuge reads from contaminated reference
--centrifuge_threads Number of threads for Centrifuge (default=12)
--skip_krona Skip the generation of Krona plots (default=false)
Polishing:
--skip_polishing Skip the Racon and Medaka polishing step (default=false)
--racon_nb Number of Racon long-read polishing iterations (default=4)
--racon_args Racon optional parameters (default="-m 8 -x -6 -g -8 -w 500")
--racon_threads Number of threads for Racon (default=4)
--medaka_threads Number of threads for Medaka (default=4)
--medaka_model Medaka model (default=r1041_e82_400bps_sup_g615)
Eukaryote and prokaryote classification:
--skip_whokaryote Skip whokaryote classification (default=false)
--whokaryote_threads Number of threads for Whokaryote (default=8)
Virus and Plasmid classification:
--skip_download_genomad_db Skip the genomad database download if it is already present locally (default=false)
--skip_genomad Skip genomad classification (default=false)
Aviary Recover MAGs:
--skip_aviary Skip aviary recover (default=false)
--aviary_threads Number of threads for Aviary (default=8)
--pplacer_threads Number of threads for Aviary (default=8)
--max_memory_aviary Maximum memory for Aviary (default=500)
--checkm_db Path to the CheckM2 database
--gtdb_path Path to the GTDB database
--eggnog_db Path to the eggnog-mapper database
Assembly quality assessment with QUAST:
--skip_quast Skip QUAST step (default=false)
--quast_threads Number of threads for QUAST (default=2)
""".stripIndent()
}
// Show help message
params.help = false
if (params.help){
helpMessage()
exit 0
}
process porechop {
cpus "${params.porechop_threads}"
tag "${sample}"
label "cpu"
label "high_memory"
publishDir "$params.outdir/$sample/1_trimming", mode: 'copy', pattern: "*.log", saveAs: { filename -> "${sample}_$filename" }
publishDir "$params.outdir/$sample/1_trimming", mode: 'copy', pattern: "*_version.txt"
publishDir "$params.outdir/$sample/1_trimming", mode: 'copy', pattern: '*fastq.gz', saveAs: { filename -> "${sample}_$filename" }
input:
tuple val(sample), path(reads), path(csv)
output:
tuple val(sample), path("trimmed.fastq.gz"), path(csv), emit: trimmed_fastq
path("porechop.log")
path("porechop_version.txt")
path("*fastq.gz")
when:
!params.skip_porechop
script:
"""
set +eu
porechop -i ${reads} -t ${params.porechop_threads} -o trimmed.fastq.gz ${params.porechop_args}
cp .command.log porechop.log
porechop --version > porechop_version.txt
"""
}
process extract_adaptive_readID {
tag "${sample}"
label "cpu"
label "high_memory"
publishDir "$params.outdir/$sample/2_adaptive", mode: 'copy', pattern: "*.log", saveAs: { filename -> "${sample}_$filename" }
input:
tuple val(sample), path(reads), path(csv)
output:
tuple val(sample), path(reads), path("adaptive_reads.txt"), path("non_adaptive_reads.txt"), emit: extracted_readID
path("extract_adaptive_readID.log")
path("*txt")
when:
!params.skip_extract_adaptive
shell:
'''
set +eu
awk -F, '$7 == "stop_receiving" {print $0}' !{csv} | cut -d"," -f5 | sort | uniq > adaptive_reads.txt
seqkit fx2tab !{reads} | awk '{print $1, $5}' - | sed 's/=/ /' | cut -d" " -f1,3 | awk '$2 > 256 {print $1}' - | sort | uniq > non_adaptive_reads.txt
cp .command.log extract_adaptive_readID.log
'''
}
process extract_adaptive_fastq {
tag "${sample}"
label "cpu"
label "high_memory"
publishDir "$params.outdir/$sample/2_adaptive", mode: 'copy', pattern: "*.log", saveAs: { filename -> "${sample}_$filename" }
publishDir "$params.outdir/$sample/2_adaptive", mode: 'copy', pattern: "*_version.txt"
publishDir "$params.outdir/$sample/2_adaptive", mode: 'copy', pattern: '*fastq', saveAs: { filename -> "${sample}_$filename" }
input:
tuple val(sample), path(reads), path(readID_adaptive), path(readID_nonadaptive)
output:
tuple val(sample),path("adaptive.fastq"),path("non_adaptive.fastq"), emit: extracted_fastq
path("extract_adaptive_fastq.log")
path("*fastq")
when:
!params.skip_extract_adaptive
shell:
'''
set +eu
seqtk subseq !{reads} !{readID_adaptive} > adaptive.fastq
seqtk subseq !{reads} !{readID_nonadaptive} > non_adaptive.fastq
cp .command.log extract_adaptive_fastq.log
'''
}
process extract_adaptive_sampling_reads {
tag "${sample}"
label "cpu"
label "high_memory"
publishDir "$params.outdir/$sample/2_adaptive", mode: 'copy', pattern: "*.log", saveAs: { filename -> "${sample}_$filename" }
publishDir "$params.outdir/$sample/2_adaptive", mode: 'copy', pattern: "*_version.txt"
publishDir "$params.outdir/$sample/2_adaptive", mode: 'copy', pattern: '*fastq', saveAs: { filename -> "${sample}_$filename" }
input:
tuple val(sample), path(reads), path(csv)
output:
tuple val(sample),path("stopreceiving.fastq"),path("unblock.fastq"),path("nodecision.fastq"), emit: extracted_fastq
path("extract_adaptive_fastq.log")
path("*nodecision.fastq")
path("unblock.fastq")
when:
!params.skip_adaptive_sampling_metrics
shell:
'''
set +eu
awk -F, '$7 == "stop_receiving" {print $0}' !{csv} | cut -d"," -f5 | sort | uniq | sort > readID_stop_receiving.txt
awk -F, '$7 == "no_decision" {print $0}' !{csv} | cut -d"," -f5 | sort | uniq | sort > readID_no_decision.txt
awk -F, '$7 == "unblock" {print $0}' !{csv} | cut -d"," -f5 | sort | uniq | sort > readID_unblock.txt
comm -12 readID_no_decision.txt readID_unblock.txt > comm_nodecision_unblock.txt
awk -v FS="[\t= ]" ' FNR==NR { a[$1]=$1; next } !($1 in a){print $0}' readID_no_decision.txt readID_unblock.txt > readID_unblock_not_no_decision.txt
cat readID_unblock_not_no_decision.txt comm_nodecision_unblock.txt | sort | uniq > readID_unblock_unique.txt
awk -v FS="[\t= ]" ' FNR==NR { a[$1]=$1; next } !($1 in a){print $0}' readID_unblock.txt readID_no_decision.txt > readID_unique_no_decision_unblock.txt
awk -v FS="[\t= ]" ' FNR==NR { a[$1]=$1; next } !($1 in a){print $0}' readID_stop_receiving.txt readID_unique_no_decision_unblock.txt > readID_no_decision_unique.txt
seqtk subseq !{reads} readID_stop_receiving.txt > stopreceiving.fastq
seqtk subseq !{reads} readID_no_decision_unique.txt > nodecision.fastq
seqtk subseq !{reads} readID_unblock_unique.txt > unblock.fastq
nb_reads=`cut -d"," -f5 !{csv} | tail -n +2 | sort | uniq | sort | wc -l`
echo -e !{sample}\\t$nb_reads >> nbReads_AS_csv.txt
cp .command.log extract_adaptive_fastq.log
'''
}
process compute_adaptive_sampling_metrics {
tag "${sample}"
label "cpu"
publishDir "$params.outdir/$sample/2_adaptive", mode: 'copy', pattern: '*txt', saveAs: { filename -> "${sample}_$filename" }
input:
tuple val(sample), path(fq_stopreceiving), path(fq_unblock), path(fq_nodecision)
output:
tuple val(sample),path("NanoStats.txt")
when:
!params.skip_adaptive_sampling_metrics
shell:
'''
set +eu
NanoComp -o \$PWD --fastq !{fq_stopreceiving} !{fq_unblock} !{fq_nodecision} -t !{params.nanocomp_threads} -n stop_receiving unblock no_decision
grep "N50" NanoStats.txt | tr -d ' ' | sed s/ReadlengthN50:// | sed s/\\.0/\\t/g >> ReadN50.txt
grep "Number" NanoStats.txt| grep reads | grep -v percentage | tr -d ' ' | sed s/Numberofreads://| sed s/\\.0/\\t/g >> NbReads.txt
grep "Median" NanoStats.txt | grep length |tr -d ' ' | sed s/Medianreadlength:// | sed s/\\.0/\\t/g >> MedianReadLength.txt
grep "Median" NanoStats.txt | grep quality |tr -s ' ' | sed s/^Median\\sread\\squality:// | sed s/\\s/\\t/g | sed s/^\\s//g >> MedianReadQuality.txt
echo !{sample} >> samples.txt
cp .command.log compute_adaptive_sampling_metrics.log
'''
}
process minimap {
cpus "${params.minimap_threads}"
tag "${sample}"
label "high_memory"
label "cpu"
publishDir "$params.outdir/$sample/3_minimap", mode: 'copy', pattern: "*.log", saveAs: { filename -> "${sample}_$filename" }
publishDir "$params.outdir/$sample/3_minimap", mode: 'copy', pattern: "*fastq", saveAs: { filename -> "${sample}_$filename" }
publishDir "$params.outdir/$sample/3_minimap", mode: 'copy', pattern: "*txt", saveAs: { filename -> "${sample}_$filename" }
input:
tuple val(sample), path(fastq_adaptive), path(fastq_non_adaptive)
output:
tuple val(sample), path("adaptive_bac.fastq"), path("non_adaptive_bac.fastq"), emit: bacterial_fastq
path("minimap.log")
path("*fastq")
path("*txt")
when:
!params.skip_remove_host_reads
shell:
'''
set +eu
module load samtools/1.13-gcc-10.3.0 seqtk/1.3-gcc-10.3.0
/scratch/project/gihcomp/sw/minimap2/minimap2 -t !{params.minimap_threads} -ax map-ont !{params.ref_genome} !{fastq_non_adaptive} > non_adaptive.sam
/scratch/project/gihcomp/sw/minimap2/minimap2 -t !{params.minimap_threads} -ax map-ont !{params.ref_genome} !{fastq_adaptive} > adaptive.sam
for type in adaptive non_adaptive; do
samtools sort -o ${type}.bam -@ !{params.minimap_threads} ${type}.sam
samtools index ${type}.bam
samtools flagstat ${type}.bam > ${type}.flagstat.txt
samtools view -S -f 4 -b ${type}.bam -o ${type}_non_host.unsorted.bam
samtools sort -o ${type}_non_host.bam -@ !{params.minimap_threads} ${type}_non_host.unsorted.bam
samtools index ${type}_non_host.bam
samtools flagstat ${type}_non_host.bam > ${type}_non_host.flagstat.txt
samtools view ${type}_non_host.bam | cut -f1 | sort | uniq > ${type}_non_host_readID.lst
done
seqtk subseq !{fastq_adaptive} adaptive_non_host_readID.lst > adaptive_bac.fastq
seqtk subseq !{fastq_non_adaptive} non_adaptive_non_host_readID.lst > non_adaptive_bac.fastq
cp .command.log minimap.log
'''
}
prefix="assembly"
prefix_lr="assembly_polished"
raconv="racon"
medakav="medaka"
process flye {
cpus "${params.flye_threads}"
tag "${sample}"
label "high_memory"
publishDir "$params.outdir/$sample/6_assembly", mode: 'copy', pattern: "*.log", saveAs: { filename -> "${sample}_$filename" }
publishDir "$params.outdir/$sample/6_assembly", mode: 'copy', pattern: "adaptive_assembly*", saveAs: { filename -> "${sample}_$filename" }
publishDir "$params.outdir/$sample/6_assembly", mode: 'copy', pattern: "non_adaptive_assembly*", saveAs: { filename -> "${sample}_$filename" }
publishDir "$params.outdir/$sample/6_assembly", mode: 'copy', pattern: "*txt", saveAs: { filename -> "${sample}_$filename" }
input:
tuple val(sample), path(fastq_adaptive_bac), path(fastq_non_adaptive_bac)
output:
tuple val(sample), path(fastq_adaptive_bac), path("adaptive_assembly_bac.fasta"), path(fastq_non_adaptive_bac), path("non_adaptive_assembly_bac.fasta"), emit: bacterial_assembly_fasta
tuple val(sample), path("adaptive_assembly_info_bac.txt"), path("adaptive_assembly_graph_bac.gfa"),path("adaptive_assembly_graph_bac.gv"), path("non_adaptive_assembly_info_bac.txt"), path("non_adaptive_assembly_graph_bac.gfa"),path("non_adaptive_assembly_graph_bac.gv"), emit: bacterial_assembly_graph
path("flye.log")
path("flye_version.txt")
when:
!params.skip_assembly
shell:
'''
set +eu
flye --nano-hq !{fastq_adaptive_bac} --threads !{params.flye_threads} --out-dir \$PWD !{params.flye_args}
if [ -f "assembly.fasta" ]; then
mv assembly.fasta adaptive_assembly_bac.fasta
mv assembly_info.txt adaptive_assembly_info_bac.txt
mv assembly_graph.gfa adaptive_assembly_graph_bac.gfa
mv assembly_graph.gv adaptive_assembly_graph_bac.gv
else
touch adaptive_assembly_bac.fasta adaptive_assembly_info_bac.txt adaptive_assembly_graph_bac.gfa adaptive_assembly_graph_bac.gv
fi
flye --nano-hq !{fastq_non_adaptive_bac} --threads !{params.flye_threads} --out-dir \$PWD !{params.flye_args}
if [ -f "assembly.fasta" ]; then
mv assembly.fasta non_adaptive_assembly_bac.fasta
mv assembly_info.txt non_adaptive_assembly_info_bac.txt
mv assembly_graph.gfa non_adaptive_assembly_graph_bac.gfa
mv assembly_graph.gv non_adaptive_assembly_graph_bac.gv
else
touch non_adaptive_assembly_bac.fasta non_adaptive_assembly_info_bac.txt non_adaptive_assembly_graph_bac.gfa non_adaptive_assembly_graph_bac.gv
fi
flye -v 2> flye_version.txt
cp .command.log flye.log
'''
}
process racon {
cpus "${params.racon_threads}"
tag "${sample}"
label "racon"
publishDir "$params.outdir/$sample/6_assembly", mode: 'copy', pattern: '*fasta', saveAs: { filename -> "${sample}_$filename"}
publishDir "$params.outdir/$sample/6_assembly", mode: 'copy', pattern: '*log', saveAs: { filename -> "${sample}_$filename" }
publishDir "$params.outdir/$sample/6_assembly", mode: 'copy', pattern: "*_version.txt"
input:
tuple val(sample), path(fastq_adaptive_bac), path(adaptive_assembly), path(fastq_non_adaptive_bac), path(non_adaptive_assembly)
output:
tuple val(sample), path(fastq_adaptive_bac), path("adaptive_${prefix}_${raconv}_${params.racon_nb}.fasta"), path(fastq_non_adaptive_bac), path("non_adaptive_${prefix}_${raconv}_${params.racon_nb}.fasta"), emit: polished_racon
path("racon.log")
path("racon_version.txt")
when:
!params.skip_polishing
script:
"""
set +eu
ln -s ${adaptive_assembly} adaptive_${prefix}_${raconv}_0.fasta
for i in `seq 1 ${params.racon_nb}`; do
ii=\$((\$i-1))
minimap2 -t ${params.racon_threads} -ax map-ont adaptive_${prefix}_${raconv}_\$ii.fasta ${fastq_adaptive_bac} > adaptive_${prefix}.gfa\$i.sam
racon ${params.racon_args} -t ${params.racon_threads} ${fastq_adaptive_bac} adaptive_${prefix}.gfa\$i.sam adaptive_${prefix}_${raconv}_\$ii.fasta --include-unpolished > adaptive_${prefix}_${raconv}_\$i.fasta
rm adaptive_${prefix}.gfa\$i.sam
done
ln -s ${non_adaptive_assembly} non_adaptive_${prefix}_${raconv}_0.fasta
for i in `seq 1 ${params.racon_nb}`; do
ii=\$((\$i-1))
minimap2 -t ${params.racon_threads} -ax map-ont non_adaptive_${prefix}_${raconv}_\$ii.fasta ${fastq_non_adaptive_bac} > non_adaptive_${prefix}.gfa\$i.sam
racon ${params.racon_args} -t ${params.racon_threads} ${fastq_non_adaptive_bac} non_adaptive_${prefix}.gfa\$i.sam non_adaptive_${prefix}_${raconv}_\$ii.fasta --include-unpolished > non_adaptive_${prefix}_${raconv}_\$i.fasta
rm non_adaptive_${prefix}.gfa\$i.sam
done
cp .command.log racon.log
racon --version > racon_version.txt
"""
}
process medaka {
cpus "${params.medaka_threads}"
tag "${sample}"
label "medaka"
publishDir "$params.outdir/$sample/6_assembly", mode: 'copy', pattern: '*fasta', saveAs: { filename -> "${sample}_$filename"}
publishDir "$params.outdir/$sample/6_assembly", mode: 'copy', pattern: '*log', saveAs: { filename -> "${sample}_$filename" }
publishDir "$params.outdir/$sample/6_assembly", mode: 'copy', pattern: "*_version.txt"
input:
tuple val(sample), path(fastq_adaptive_bac), path(adaptive_draft), path(fastq_non_adaptive_bac), path(non_adaptive_draft)
output:
tuple val(sample), path(fastq_adaptive_bac), path ("adaptive_flye_polished.fasta"), path(fastq_non_adaptive_bac), path ("non_adaptive_flye_polished.fasta"), emit: polished_medaka
path("medaka.log")
path("medaka_version.txt")
when:
!params.skip_polishing
script:
"""
set +eu
medaka_consensus -i ${fastq_adaptive_bac} -d ${adaptive_draft} -o \$PWD -t ${params.medaka_threads} -m ${params.medaka_model}
rm consensus_probs.hdf calls_to_draft.bam calls_to_draft.bam.bai
if [ -f "consensus.fasta" ]; then
mv consensus.fasta adaptive_flye_polished.fasta
else
touch adaptive_flye_polished.fasta
fi
medaka_consensus -i ${fastq_adaptive_bac} -d ${non_adaptive_draft} -o \$PWD -t ${params.medaka_threads} -m ${params.medaka_model}
rm consensus_probs.hdf calls_to_draft.bam calls_to_draft.bam.bai
if [ -f "consensus.fasta" ]; then
mv consensus.fasta non_adaptive_flye_polished.fasta
else
touch non_adaptive_flye_polished.fasta
fi
cp .command.log medaka.log
medaka --version > medaka_version.txt
"""
}
process centrifuge_download_db {
cpus 1
label "high_memory"
publishDir "$params.outdir/centrifuge_database", mode: 'copy', pattern: "*.cf"
input:
val(db)
output:
tuple path("*.1.cf"), path("*.2.cf"), path("*.3.cf"), path("*.4.cf"), emit: centrifuge_db
when:
!params.skip_download_centrifuge_db
script:
"""
echo ${db}
wget ${db}
tar -xvf nt_2018_3_3.tar.gz
"""
}
process centrifuge {
cpus "${params.centrifuge_threads}"
tag "${sample}"
label "very_high_memory"
publishDir "$params.outdir/$sample/4_centrifuge", mode: 'copy', pattern: "*.tsv", saveAs: { filename -> "${sample}_$filename" }
publishDir "$params.outdir/$sample/4_centrifuge", mode: 'copy', pattern: "*.log", saveAs: { filename -> "${sample}_$filename" }
input:
tuple val(sample), path(fastq_adaptive_bac), path(fastq_non_adaptive_bac), path(db1), path(db2), path(db3), path(db4)
output:
tuple val(sample), path(fastq_adaptive_bac), path("adaptive_centrifuge_species_report.tsv"), path(fastq_non_adaptive_bac), path("non_adaptive_centrifuge_species_report.tsv"), emit: bacterial_fastq
tuple val(sample), path("adaptive_centrifuge_species_report.tsv"), path("non_adaptive_centrifuge_species_report.tsv"), emit: centrifuge_species_report
tuple val(sample), path("adaptive_centrifuge_report.tsv"), path("non_adaptive_centrifuge_report.tsv"), emit: centrifuge_report
path("centrifuge.log")
when:
!params.skip_centrifuge
script:
"""
centrifuge -x nt -U ${fastq_adaptive_bac} -S adaptive_centrifuge_species_report.tsv --report-file adaptive_centrifuge_report.tsv --threads ${params.centrifuge_threads}
centrifuge -x nt -U ${fastq_non_adaptive_bac} -S non_adaptive_centrifuge_species_report.tsv --report-file non_adaptive_centrifuge_report.tsv --threads ${params.centrifuge_threads}
cp .command.log centrifuge.log
"""
}
process remove_centrifuge_contaminated {
tag "${sample}"
//label "very_high_memory"
publishDir "$params.outdir/$sample/5_centrifuge_bac_reads", mode: 'copy', pattern: "*.lst", saveAs: { filename -> "${sample}_$filename" }
publishDir "$params.outdir/$sample/5_centrifuge_bac_reads", mode: 'copy', pattern: "*.fastq", saveAs: { filename -> "${sample}_$filename" }
publishDir "$params.outdir/$sample/5_centrifuge_bac_reads", mode: 'copy', pattern: "*.log", saveAs: { filename -> "${sample}_$filename" }
input:
tuple val(sample), path(fastq_adaptive_bac), path("adaptive_centrifuge_species_report.tsv"), path(fastq_non_adaptive_bac), path("non_adaptive_centrifuge_species_report.tsv")
output:
tuple val(sample), path("adaptive_centrifuge_bac_readID.lst"), path("non_adaptive_centrifuge_bac_readID.lst"), path("adaptive_bacterial.fastq"), path("non_adaptive_bacterial.fastq"), emit: bac_fastq_readID
tuple val(sample), path("adaptive_centrifuge_species_report_filtered.tsv"), path("non_adaptive_centrifuge_species_report_filtered.tsv"), emit: input_krona
tuple val(sample), path("adaptive_bacterial.fastq"), path("non_adaptive_bacterial.fastq"), emit: bac_fastq
when:
!skip_centrifuge_remove_contaminated
shell:
'''
set +eu
awk '$3 ~ !{params.centrifuge_reference_tax_ID}' !{"adaptive_centrifuge_species_report.tsv"} | cut -f1 | sort | uniq > adaptive_centrifuge_host_readID.lst
awk '$3 !~ !{params.centrifuge_reference_tax_ID}' !{"adaptive_centrifuge_species_report.tsv"} | cut -f1 | sort | uniq > adaptive_centrifuge_bac_host_readID.lst
awk -v FS="[\t= ]" ' FNR==NR { a[$1]=$1; next } !($1 in a){print $0}' adaptive_centrifuge_host_readID.lst adaptive_centrifuge_bac_host_readID.lst > adaptive_centrifuge_bac_readID.lst
awk '$3 ~ !{params.centrifuge_reference_tax_ID}' !{"non_adaptive_centrifuge_species_report.tsv"} | cut -f1 | sort | uniq > non_adaptive_centrifuge_host_readID.lst
awk '$3 !~ !{params.centrifuge_reference_tax_ID}' !{"non_adaptive_centrifuge_species_report.tsv"} | cut -f1 | sort | uniq > non_adaptive_centrifuge_bac_host_readID.lst
awk -v FS="[\t= ]" ' FNR==NR { a[$1]=$1; next } !($1 in a){print $0}' non_adaptive_centrifuge_host_readID.lst non_adaptive_centrifuge_bac_host_readID.lst > non_adaptive_centrifuge_bac_readID.lst
seqtk subseq !{fastq_adaptive_bac} adaptive_centrifuge_bac_readID.lst > adaptive_bacterial.fastq
seqtk subseq !{fastq_non_adaptive_bac} non_adaptive_centrifuge_bac_readID.lst > non_adaptive_bacterial.fastq
awk -v FS="[\t= ]" ' FNR==NR { a[$1]=$1; next } !($1 in a){print $0}' adaptive_centrifuge_host_readID.lst adaptive_centrifuge_species_report.tsv > adaptive_centrifuge_species_report_filtered.tsv
awk -v FS="[\t= ]" ' FNR==NR { a[$1]=$1; next } !($1 in a){print $0}' non_adaptive_centrifuge_host_readID.lst non_adaptive_centrifuge_species_report.tsv > non_adaptive_centrifuge_species_report_filtered.tsv
cp .command.log remove_centrifuge_contaminated.log
'''
}
process krona {
cpus 1
tag "${sample}"
publishDir "$params.outdir/$sample/5_centrifuge_bac_reads", mode: 'copy', pattern: "*krona.html", saveAs: { filename -> "${sample}_$filename" }
publishDir "$params.outdir/$sample/5_centrifuge_bac_reads", mode: 'copy', pattern: "*.log", saveAs: { filename -> "${sample}_$filename" }
input:
tuple val(sample), path(adaptive_species_report), path(non_adaptive_species_report), path(krona_database)
output:
tuple val(sample), path("adaptive_centrifuge_taxonomy.krona.html"), path("non_adaptive_centrifuge_taxonomy.krona.html"), emit: krona_html
path("krona.log")
when:
!params.skip_krona | !params.skip_centrifuge
script:
"""
set +eu
cat ${adaptive_species_report} | cut -f 1,3 > adaptive_centrifuge_species_report.krona
cat ${non_adaptive_species_report} | cut -f 1,3 > non_adaptive_centrifuge_species_report.krona
ktImportTaxonomy adaptive_centrifuge_species_report.krona -o adaptive_centrifuge_taxonomy.krona.html -tax \$PWD
ktImportTaxonomy non_adaptive_centrifuge_species_report.krona -o non_adaptive_centrifuge_taxonomy.krona.html -tax \$PWD
cp .command.log krona.log
"""
}
process whokaryote {
cpus "${params.whokaryote_threads}"
tag "${sample}"
publishDir "$params.outdir/$sample/7_whokaryote", mode: 'copy', pattern: "{*sv}", saveAs: { filename -> "${sample}_$filename" }
publishDir "$params.outdir/$sample/7_whokaryote", mode: 'copy', saveAs: { filename -> "${sample}_$filename" }
input:
tuple val(sample), path(fastq_adaptive_bac), path(adaptive_assembly), path(fastq_non_adaptive_bac), path(non_adaptive_assembly)
output:
tuple val(sample), path("adaptive_whokaryote_predictions_T.tsv"), path("non_adaptive_whokaryote_predictions_T.tsv"), emit: whokaryote_prediction
tuple val(sample), path("adaptive_featuretable_predictions_T.tsv"), path("adaptive_featuretable.csv"), path("adaptive_contigs_genes.gff"), path("adaptive_contigs_proteins.faa"), path("adaptive_tiara_pred.txt"), path("non_adaptive_featuretable_predictions_T.tsv"), path("non_adaptive_featuretable.csv"), path("non_adaptive_contigs_genes.gff"), path("non_adaptive_contigs_proteins.faa"), path("non_adaptive_tiara_pred.txt"), emit: whokaryote_results
path("whokaryote.log")
when:
!params.skip_whokaryote | !params.skip_assembly
script:
"""
set +eu
whokaryote.py --contigs ${adaptive_assembly} --outdir \$PWD --threads ${params.whokaryote_threads}
mv whokaryote_predictions_T.tsv adaptive_whokaryote_predictions_T.tsv
mv featuretable_predictions_T.tsv adaptive_featuretable_predictions_T.tsv
mv featuretable.csv adaptive_featuretable.csv
mv contigs_genes.gff adaptive_contigs_genes.gff
mv contigs_proteins.faa adaptive_contigs_proteins.faa
mv tiara_pred.txt adaptive_tiara_pred.txt
mv contigs5000.fasta adaptive_contigs5000.fasta
whokaryote.py --contigs ${non_adaptive_assembly} --outdir \$PWD --threads ${params.whokaryote_threads}
mv whokaryote_predictions_T.tsv non_adaptive_whokaryote_predictions_T.tsv
mv featuretable_predictions_T.tsv non_adaptive_featuretable_predictions_T.tsv
mv featuretable.csv non_adaptive_featuretable.csv
mv contigs_genes.gff non_adaptive_contigs_genes.gff
mv contigs_proteins.faa non_adaptive_contigs_proteins.faa
mv tiara_pred.txt non_adaptive_tiara_pred.txt
cp .command.log whokaryote.log
"""
}
process download_genomad_db {
cpus 1
label "high_memory"
publishDir "$params.outdir/genomad_database", mode: 'copy'
input:
val(db)
output:
path("genomad_db/*"), emit: genomad_db
when:
!params.skip_download_genomad_db | !params.skip_assembly
script:
"""
echo ${db}
wget ${db}
tar -xvf genomad_db_v1.5.tar.gz
"""
}
process genomad {
cpus 1
tag "${sample}"
label "high_memory"
//publishDir "$params.outdir/$sample/8_genomad", mode: 'copy', pattern: "*.tsv", saveAs: { filename -> "${sample}_$filename" }
publishDir "$params.outdir/$sample/8_genomad", mode: 'copy', saveAs: { filename -> "${sample}_$filename" }
input:
tuple val(sample), path(fastq_adaptive_bac), path(adaptive_assembly), path(fastq_non_adaptive_bac), path(non_adaptive_assembly), path(genomad_db)
output:
path("*_aggregated_classification/*_aggregated_classification.tsv") , emit: aggregated_classification
path("*_annotate/*_taxonomy.tsv") , emit: taxonomy
path("*_find_proviruses/*_provirus.tsv") , emit: provirus
//path("*_score_calibration/*_compositions.tsv") , emit: compositions
//path("*_score_calibration/*_calibrated_aggregated_classification.tsv") , emit: calibrated_classification
path("*_summary/*_plasmid.fna") , emit: plasmid_fasta
path("*_summary/*_plasmid_genes.tsv") , emit: plasmid_genes
path("*_summary/*_plasmid_proteins.faa") , emit: plasmid_proteins
path("*_summary/*_plasmid_summary.tsv") , emit: plasmid_summary
path("*_summary/*_virus.fna") , emit: virus_fasta
path("*_summary/*_virus_genes.tsv") , emit: virus_genes
path("*_summary/*_virus_proteins.faa") , emit: virus_proteins
path("*_summary/*_virus_summary.tsv") , emit: virus_summary
//path "versions.yml" , emit: versions
when:
!params.skip_genomad | !params.skip_assembly
script:
"""
if [ -s ${adaptive_assembly} ] && [ -s ${non_adaptive_assembly} ] ; then
genomad end-to-end --cleanup --splits 4 ${adaptive_assembly} \$PWD ${genomad_db}
genomad end-to-end --cleanup --splits 4 ${non_adaptive_assembly} \$PWD ${genomad_db}
elif [ -s ${adaptive_assembly} ] ; then
genomad end-to-end --cleanup --splits 4 ${adaptive_assembly} \$PWD ${genomad_db}
elif [ -s ${non_adaptive_assembly} ] ; then
genomad end-to-end --cleanup --splits 4 ${non_adaptive_assembly} \$PWD ${genomad_db}
else
echo "both non_adaptive and adaptive assembly files are empty, so genomad was not run"
fi
"""
}
process aviary_recover {
cpus "${params.aviary_threads}"
tag "${sample}"
label "high_memory"
//publishDir "$params.outdir/$sample/9_aviary", mode: 'copy', pattern: "{*sv}", saveAs: { filename -> "${sample}_$filename" }
publishDir "$params.outdir/$sample/9_aviary", mode: 'copy'
input:
tuple val(sample), path(fastq_adaptive_bac), path(adaptive_assembly), path(fastq_non_adaptive_bac), path(non_adaptive_assembly)
output:
path("${sample}_adaptive/bins/*"), emit: bins
path("${sample}_adaptive/data/*"), emit: data
path("${sample}_adaptive/diversity/*"), emit: diversity
path("${sample}_adaptive/benchmarks/*"), emit: benchmarks
path("${sample}_non_adaptive/bins/*"), emit: non_adaptive_bins
path("${sample}_non_adaptive/data/*"), emit: non_adaptive_data
path("${sample}_non_adaptive/diversity/*"), emit: non_adaptive_diversity
path("${sample}_non_adaptive/benchmarks/*"), emit: non_adaptive_benchmarks
path("aviary.log")
when:
!params.skip_aviary | !params.skip_assembly
script:
"""
set +eu
aviary recover \
--assembly ${adaptive_assembly} \
--longreads ${fastq_adaptive_bac} \
--longread-type ont_hq \
--n-cores ${params.aviary_threads} \
--skip-binners semibin \
--max_threads ${params.aviary_threads} \
--pplacer_threads ${params.pplacer_threads} \
--max_memory ${params.max_memory_aviary} \
--checkm2-db-path ${params.checkm_db} \
--gtdb-path ${params.gtdb_path} \
--eggnog-db-path ${params.eggnog_db} \
--workflow recover_mags \
--output \$PWD
mkdir ${sample}_adaptive
mv data/ bins/ benchmarks/ diversity/ config.yaml ${sample}_adaptive/
aviary recover \
--assembly ${non_adaptive_assembly} \
--longreads ${fastq_non_adaptive_bac} \
--longread-type ont_hq \
--n-cores ${params.aviary_threads} \
--skip-binners semibin \
--max_threads ${params.aviary_threads} \
--pplacer_threads ${params.pplacer_threads} \
--max_memory ${params.max_memory_aviary} \
--checkm2-db-path ${params.checkm_db} \
--gtdb-path ${params.gtdb_path} \
--eggnog-db-path ${params.eggnog_db} \
--workflow recover_mags \
--output \$PWD
mkdir ${sample}_non_adaptive
mv data/ bins/ benchmarks/ diversity/ config.yaml ${sample}_non_adaptive/
cp .command.log aviary.log
"""
}
process quast {
cpus "${params.quast_threads}"
tag "${sample}"
publishDir "$params.outdir/$sample/10_quast", mode: 'copy'
//publishDir "$params.outdir/$sample/10_quast", mode: 'copy', pattern: "{*sv}", saveAs: { filename -> "${sample}_$filename" }
input:
tuple val(sample), path(fastq_adaptive_bac), path(adaptive_assembly), path(fastq_non_adaptive_bac), path(non_adaptive_assembly)
output:
path("${sample}/*"), emit: quast_results
path("quast.log")
when:
!params.skip_quast | !params.skip_assembly
script:
"""
metaquast.py -l ${sample}_AS,${sample}_NAS ${adaptive_assembly} ${non_adaptive_assembly} --max-ref-number 0 --threads ${params.quast_threads} -o \$PWD/${sample}
cp .command.log quast.log
"""
}
workflow {
ch_centrifuge_db=Channel.value( "${params.centrifuge_db}")
ch_centrifuge_db.view()
ch_genomad_db=Channel.value( "${params.genomad_db}" )
ch_genomad_db.view()
Channel.fromPath( "${params.samplesheet}", checkIfExists:true )
.splitCsv(header:true, sep:',')
.map { row -> tuple(row.sample_id, file(row.fastq, checkIfExists: true), file(row.csv, checkIfExists: true)) }
.set { ch_samplesheet }
ch_samplesheet.view()
if (!params.skip_porechop) {
porechop(ch_samplesheet)
extract_adaptive_readID(porechop.out.trimmed_fastq)
} else if (params.skip_porechop) {
extract_adaptive_readID(ch_samplesheet)
}
extract_adaptive_fastq(extract_adaptive_readID.out.extracted_readID)
if (!params.skip_adaptive_sampling_metrics) {
extract_adaptive_sampling_reads(ch_samplesheet)
compute_adaptive_sampling_metrics(extract_adaptive_sampling_reads.out.extracted_fastq)
}
minimap(extract_adaptive_fastq.out.extracted_fastq)
if (!params.skip_centrifuge) {
if (!params.skip_download_centrifuge_db) {
centrifuge_download_db(ch_centrifuge_db)
centrifuge(minimap.out.bacterial_fastq.combine(centrifuge_download_db.out.centrifuge_db))
} else if (params.skip_download_centrifuge_db) {
ch_centrifuge_db=Channel.fromPath( "${params.outdir}/centrifuge_database/*.cf" ).collect()
centrifuge(minimap.out.bacterial_fastq.combine(ch_centrifuge_db))
}
remove_centrifuge_contaminated(centrifuge.out.bacterial_fastq)
if (!params.skip_krona) {
ch_krona_db=Channel.value( "${params.krona_db}")
krona(remove_centrifuge_contaminated.out.input_krona.combine(ch_krona_db))
}
if (!params.skip_assembly) {
flye(remove_centrifuge_contaminated.out.bac_fastq)
if (!params.skip_polishing) {
racon(flye.out.bacterial_assembly_fasta)
medaka(racon.out.polished_racon)
if (!params.skip_whokaryote) {
whokaryote(medaka.out.polished_medaka)
}
if (!params.skip_genomad) {
if (!params.skip_download_genomad_db) {
download_genomad_db(ch_genomad_db)
genomad(medaka.out.polished_medaka.combine(download_genomad_db.out.genomad_db))
} else if (params.skip_download_genomad_db) {
ch_genomad_db=Channel.fromPath( "${params.outdir}/genomad_database/genomad_db/" ).collect()
genomad(medaka.out.polished_medaka.combine(ch_genomad_db))
}
}
if (!params.skip_aviary) {
aviary_recover(medaka.out.polished_medaka)
}
if (!params.skip_quast) {
quast(medaka.out.polished_medaka)
}
} else if (params.skip_polishing) {
if (!params.skip_whokaryote) {
whokaryote(flye.out.bacterial_assembly_fasta)
}
if (!params.skip_genomad) {
if (!params.skip_download_genomad_db) {
download_genomad_db(ch_genomad_db)
genomad(flye.out.bacterial_assembly_fasta.combine(download_genomad_db.out.genomad_db))
} else if (params.skip_download_genomad_db) {
ch_genomad_db=Channel.fromPath( "${params.outdir}/genomad_database/genomad_db/" ).collect()
genomad(flye.out.bacterial_assembly_fasta.combine(ch_genomad_db)) }
}
if (!params.skip_aviary) {
aviary_recover(flye.out.bacterial_assembly_fasta)
}
if (!params.skip_quast) {
quast(flye.out.bacterial_assembly_fasta)
}
}
}
} else if (params.skip_centrifuge) {
if (!params.skip_assembly) {
flye(minimap.out.bacterial_fastq)
if (!params.skip_polishing) {
racon(flye.out.bacterial_assembly_fasta)
medaka(racon.out.polished_racon)
if (!params.skip_whokaryote) {
whokaryote(medaka.out.polished_medaka)
}
if (!params.skip_download_genomad_db) {
download_genomad_db(ch_genomad_db)
genomad(medaka.out.polished_medaka.combine(download_genomad_db.out.genomad_db))
} else if (params.skip_download_genomad_db) {
ch_genomad_db=Channel.fromPath( "${params.outdir}/genomad_database/genomad_db/" ).collect() }
genomad(medaka.out.polished_medaka.combine(download_genomad_db.out.genomad_db))
if (!params.skip_aviary) {
aviary_recover(medaka.out.polished_medaka)
}
if (!params.skip_quast) {
quast(medaka.out.polished_medaka)
}
} else if (params.skip_polishing) {
if (!params.skip_whokaryote) {
whokaryote(flye.out.bacterial_assembly_fasta)
}
if (!params.skip_download_genomad_db) {
download_genomad_db(ch_genomad_db)
genomad(flye.out.bacterial_assembly_fasta.combine(download_genomad_db.out.genomad_db))
} else if (params.skip_download_genomad_db) {
ch_genomad_db=Channel.fromPath( "${params.outdir}/genomad_database/genomad_db/" ).collect()
genomad(flye.out.bacterial_assembly_fasta.combine(ch_genomad_db)) }
if (!params.skip_aviary) {
aviary_recover(flye.out.bacterial_assembly_fasta)
}
if (!params.skip_quast) {
quast(flye.out.bacterial_assembly_fasta)
}
}
}
}
}