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main.nf
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#!/usr/bin/env nextflow
/*
vim: syntax=groovy
-*- mode: groovy;-*-
==============================================================================
B A B S - A T A C S E Q P A I R E D - E N D B E S T P R A C T I C E
==============================================================================
ATACSeq Analysis Pipeline For Paired-End Illumina Samples.
Started 11th May 2018.
#### Homepage / Documentation
https://github.com/crickbabs/BABS-ATACSeqPE
#### Authors
Harshil Patel <[email protected]>
Philip East <[email protected]>
Nourdine Bah <[email protected]>
-------------------------------------------------------------------------------
*/
///////////////////////////////////////////////////////////////////////////////
///////////////////////////////////////////////////////////////////////////////
/* -- -- */
/* -- PARAMETERS -- */
/* -- -- */
///////////////////////////////////////////////////////////////////////////////
///////////////////////////////////////////////////////////////////////////////
def helpMessage() {
log.info"""
======================================================
BABS ATACSeq Paired-End Pipeline v${params.version}
======================================================
Usage:
The typical command for running the pipeline is as follows:
nextflow run main.nf --design design.csv --genome hg19 -profile conda
Mandatory arguments:
--design Comma separted file containing information about the samples in the experiment (see README.md)
--genome Genome shortname (e.g. hg19)
-profile Hardware config to use i.e. conda, babs_modules, standard
References: If not specified in the configuration file or you wish to overwrite any of the reference parameters
--fasta Path to Fasta reference file containing all chromosomes/contigs
--gtf Path to GTF file
--mito_name Name of Mitochondrial chomosome in genome fasta (e.g. chrM)
--bwa_index Path to BWA index
--genome_mask BED file specifying target regions for analysis. This excludes problematic genomic loci
--macs_genome_size Effective genome size parameter required by MACS2
Trimming options:
--adapter 3' adapter sequence trimmed by cutadapt (default: CTGTCTCTTATA)
Output options:
--outdir The output directory where the results will be saved (default: './results')
Other options:
-name Name for the pipeline run. If not specified, Nextflow will automatically generate a random mnemonic.
""".stripIndent()
}
// SHOW HELP MESSAGE
params.help = false
if (params.help){
helpMessage()
exit 0
}
///////////////////////////////////////////////////////////////////////////////
///////////////////////////////////////////////////////////////////////////////
/* -- -- */
/* -- SET UP CONFIGURATION VARIABLES -- */
/* -- -- */
///////////////////////////////////////////////////////////////////////////////
///////////////////////////////////////////////////////////////////////////////
////////////////////////////////////////////////////
/* -- DEFAULT PARAMETER VALUES -- */
////////////////////////////////////////////////////
params.design = false
params.genome = false
params.profile = false
params.fasta = params.genome ? params.genomes[ params.genome ].fasta ?: false : false
params.gtf = params.genome ? params.genomes[ params.genome ].gtf ?: false : false
params.mito_name = params.genome ? params.genomes[ params.genome ].mito_name ?: false : false
params.bwa_index = params.genome ? params.genomes[ params.genome ].bwa_index ?: false : false
params.genome_mask = params.genome ? params.genomes[ params.genome ].genome_mask ?: false : false
params.macs_genome_size = params.genome ? params.genomes[ params.genome ].macs_genome_size ?: false : false
params.adapter = false
params.outdir = './results'
params.outdir_abspath = new File(params.outdir).getCanonicalPath().toString()
params.name = false
params.project = false
params.multiqc_config = "$baseDir/conf/multiqc_config.yaml"
params.fastqscreen_config = "$baseDir/conf/fastq_screen.conf.txt"
params.bamtools_filter_pe_config = "$baseDir/conf/bamtools_filter_pe.json"
// PRESET ADAPTER TRIMMING OPTION
adapter_seq = 'CTGTCTCTTATA'
if (params.adapter){
adapter_seq = params.adapter
}
// SPECIFY THE RUN NAME
custom_runName = params.name
if( !(workflow.runName ==~ /[a-z]+_[a-z]+/) ){
custom_runName = workflow.runName
}
output_docs = file("$baseDir/docs/output.md")
////////////////////////////////////////////////////
/* -- CONFIG FILES -- */
////////////////////////////////////////////////////
multiqc_config = file(params.multiqc_config)
if( !multiqc_config.exists() ) exit 1, "MultiQC config file not found: ${params.multiqc_config}"
bamtools_filter_pe_config = file(params.bamtools_filter_pe_config)
if( !bamtools_filter_pe_config.exists() ) exit 1, "BamTools filter config file not found: ${params.bamtools_filter_pe_config}"
fastqscreen_config = file(params.fastqscreen_config)
////////////////////////////////////////////////////
/* -- CHECK INPUT FILES -- */
////////////////////////////////////////////////////
if( params.fasta && file(params.fasta).exists() ){
Channel
.fromPath(params.fasta)
.into { fasta_index_ch;
fasta_markdup_collectmetrics_ch;
fasta_merge_replicate_macs2_homer_ch;
fasta_merge_replicate_macs2_merge_peaks_homer_ch;
fasta_merge_sample_macs2_homer_ch;
fasta_merge_sample_macs2_merge_peaks_homer_ch;
fasta_igv_ch }
} else {
exit 1, "Genome fasta file not found: ${params.fasta}"
}
if( params.gtf && file(params.gtf).exists() ){
Channel
.fromPath(params.gtf)
.into { gtf_merge_replicate_macs2_homer_ch;
gtf_merge_replicate_macs2_merge_peaks_homer_ch;
gtf_merge_sample_macs2_homer_ch;
gtf_merge_sample_macs2_merge_peaks_homer_ch;
gtf_igv_ch }
} else {
exit 1, "GTF file not found: ${params.gtf}"
}
if( params.bwa_index && file("${params.bwa_index}.amb").exists() ) {
Channel
.fromPath("${params.bwa_index}*.{amb,ann,bwt,pac,sa}")
.into { bwa_index_bwa_aln_r1_ch;
bwa_index_bwa_aln_r2_ch;
bwa_index_bwa_sampe_ch }
} else {
exit 1, "BWA indices not found: ${params.bwa_index}"
}
if( params.design && file(params.design).exists() ){
Channel
.fromPath(params.design)
.splitCsv(header:true, sep:',')
.map { row -> [ [row.sample,"R"+row.replicate,"L"+row.run].join("_"),
row.sample,
row.replicate,
row.run,
file(row.fastq_1),
file(row.fastq_2) ] }
.into { design_replicates_exist_ch;
design_multiple_samples_ch;
design_raw_fastqc_ch;
design_raw_fastqscreen_ch;
design_cutadapt_ch }
} else {
exit 1, "Design file not found: ${params.design}"
}
///////////////////////////////////////////////////////////////////////////////
///////////////////////////////////////////////////////////////////////////////
/* -- -- */
/* -- HEADER LOG INFO -- */
/* -- -- */
///////////////////////////////////////////////////////////////////////////////
///////////////////////////////////////////////////////////////////////////////
log.info "======================================================"
log.info "BABS ATACSeq Paired-End Pipeline v${params.version}"
log.info "======================================================"
def summary = [:]
summary['Run name'] = custom_runName ?: workflow.runName
summary['Design file'] = params.design
summary['Genome version'] = params.genome
summary['Genome fasta file'] = params.fasta
summary['GTF annotation file'] = params.gtf
summary['Mitochondrial contig'] = params.mito_name
summary['BWA index'] = params.bwa_index
summary['Genome masked BED file'] = params.genome_mask
summary['MACS2 genome size'] = params.macs_genome_size
summary['Adapter sequence'] = adapter_seq
summary['Max memory'] = params.max_memory
summary['Max CPUs'] = params.max_cpus
summary['Max time'] = params.max_time
summary['Output directory'] = params.outdir
summary['Output directory path'] = params.outdir_abspath
summary['Working directory'] = workflow.workDir
summary['Current home'] = "$HOME"
summary['Current user'] = "$USER"
summary['Current path'] = "$PWD"
summary['Script directory'] = workflow.projectDir
summary['Config profile'] = workflow.profile
if(workflow.revision) summary['Pipeline release'] = workflow.revision
if(params.project) summary['BABS project'] = params.project
log.info summary.collect { k,v -> "${k.padRight(26)}: $v" }.join("\n")
log.info "======================================================"
// CHECK THAT NEXTFLOW VERSION IS UP TO DATE ENOUGH
try {
if( ! nextflow.version.matches(">= $params.nf_required_version") ){
throw GroovyException('Nextflow version too old')
}
} catch (all) {
log.error "============================================================\n" +
" Nextflow version $params.nf_required_version required! You are running v$workflow.nextflow.version.\n" +
" Pipeline execution will continue, but things may break.\n" +
" Please run `nextflow self-update` to update Nextflow.\n" +
"============================================================"
}
///////////////////////////////////////////////////////////////////////////////
///////////////////////////////////////////////////////////////////////////////
/* -- -- */
/* -- GLOBAL VARIABLES -- */
/* -- -- */
///////////////////////////////////////////////////////////////////////////////
///////////////////////////////////////////////////////////////////////////////
// BOOLEAN VALUE FOR REPLICATES EXISTING IN DESIGN
replicates_exist = design_replicates_exist_ch.map { it -> it[2].toInteger() }
.flatten()
.max()
.val > 1
// BOOLEAN VALUE FOR MULTIPLE SAMPLES EXISTING IN DESIGN
multiple_samples = design_multiple_samples_ch.map { it -> it[1] }
.flatten()
.unique()
.count()
.val > 1
///////////////////////////////////////////////////////////////////////////////
///////////////////////////////////////////////////////////////////////////////
/* -- -- */
/* -- PREPARE ANNOTATION FILES -- */
/* -- -- */
///////////////////////////////////////////////////////////////////////////////
///////////////////////////////////////////////////////////////////////////////
// CREATE FAIDX FOR REFERENCE GENOME
// CREATE CHROMOSOME SIZES FILE FOR BEDTOOLS
// CREATE BED FILE WITHOUT MASKED REGIONS & MITOCHONDRIAL CONTIG FOR SAMTOOLS FILTERING
process prep_genome {
publishDir "${params.outdir}/genome", mode: 'copy'
input:
file fasta from fasta_index_ch
output:
file "*.fai" into prep_genome_index_ch
file "*.sizes" into prep_genome_sizes_replicate_bedgraph_ch,
prep_genome_sizes_sample_bedgraph_ch,
prep_genome_sizes_replicate_bigwig_ch,
prep_genome_sizes_sample_bigwig_ch
file "*.include_regions.bed" into prep_genome_bed_filter_bam_ch
script:
if (file(params.genome_mask).exists()) {
"""
samtools faidx ${fasta}
cut -f 1,2 ${fasta}.fai > ${fasta}.sizes
sortBed -i ${params.genome_mask} -g ${fasta}.sizes | complementBed -i stdin -g ${fasta}.sizes > ${fasta}.bed
awk '\$1 != "${params.mito_name}"' ${fasta}.bed > ${fasta}.include_regions.bed
"""
} else {
"""
samtools faidx ${fasta}
cut -f 1,2 ${fasta}.fai > ${fasta}.sizes
awk 'BEGIN{OFS="\t"}{print \$1, '0' , \$2}' ${fasta}.sizes > ${fasta}.bed
awk '\$1 != "${params.mito_name}"' ${fasta}.bed > ${fasta}.include_regions.bed
"""
}
}
///////////////////////////////////////////////////////////////////////////////
///////////////////////////////////////////////////////////////////////////////
/* -- -- */
/* -- FASTQ QC -- */
/* -- -- */
///////////////////////////////////////////////////////////////////////////////
///////////////////////////////////////////////////////////////////////////////
// RUN FASTQC ON RAW FASTQ FILES
process raw_fastqc {
tag "$sampleid"
label 'mediumcpu'
publishDir "${params.outdir}/qc/fastqc/raw", mode: 'copy',
saveAs: {filename -> filename.endsWith(".zip") ? "zip/$filename" : "$filename"}
input:
set val(sampleid), val(sample), val(replicate), val(run), file(fastq_1), file(fastq_2) from design_raw_fastqc_ch
output:
set val(sampleid), file("*.{zip,html}") into raw_fastqc_ch
script:
"""
ln -s ${fastq_1} ${sampleid}_1.fastq.gz
ln -s ${fastq_2} ${sampleid}_2.fastq.gz
fastqc --outdir ./ --threads ${task.cpus} ${sampleid}_1.fastq.gz
fastqc --outdir ./ --threads ${task.cpus} ${sampleid}_2.fastq.gz
"""
}
// RUN FASTQSCREEN ON RAW FASTQ FILES
process raw_fastqscreen {
tag "$sampleid"
label 'mediumcpu'
publishDir "${params.outdir}/qc/fastq_screen", mode: 'copy',
saveAs: {filename ->
if (filename.endsWith(".txt")) "txt/$filename"
else if (filename.endsWith(".png")) "png/$filename"
else "$filename"
}
input:
set val(sampleid), val(sample), val(replicate), val(run), file(fastq_1), file(fastq_2) from design_raw_fastqscreen_ch
output:
set val(sampleid), file("*.{png,txt,html}") into raw_fastqscreen_ch
when:
params.fastqscreen_config
script:
"""
ln -s ${fastq_1} ${sampleid}_1.fastq.gz
ln -s ${fastq_2} ${sampleid}_2.fastq.gz
fastq_screen --outdir ./ \\
--conf ${fastqscreen_config} \\
--threads ${task.cpus} \\
${sampleid}_1.fastq.gz
fastq_screen --outdir ./ \\
--conf ${fastqscreen_config} \\
--threads ${task.cpus} \\
${sampleid}_2.fastq.gz
"""
}
///////////////////////////////////////////////////////////////////////////////
///////////////////////////////////////////////////////////////////////////////
/* -- -- */
/* -- ADAPTER TRIMMING -- */
/* -- -- */
///////////////////////////////////////////////////////////////////////////////
///////////////////////////////////////////////////////////////////////////////
// RUN CUTADAPT ON RAW FASTQ FILES
process cutadapt {
tag "$sampleid"
publishDir "${params.outdir}/qc/cutadapt", mode: 'copy',
saveAs: {filename ->
if (filename.endsWith(".log")) "$filename"
else null
}
input:
set val(sampleid), val(sample), val(replicate), val(run), file(fastq_1), file(fastq_2) from design_cutadapt_ch
output:
set val(sampleid), file('*.trim.fastq.gz') into cutadapt_fastqc_ch,
cutadapt_bwa_aln_r1_ch,
cutadapt_bwa_aln_r2_ch,
cutadapt_bwa_sai_to_sam_ch
set val(sampleid), file('*.log') into cutadapt_log_ch
script:
"""
ln -s ${fastq_1} ${sampleid}_1.fastq.gz
ln -s ${fastq_2} ${sampleid}_2.fastq.gz
cutadapt -a ${adapter_seq} \\
-A ${adapter_seq} \\
--minimum-length=25 \\
--quality-cutoff=20 \\
-o ${sampleid}_1.trim.fastq.gz \\
-p ${sampleid}_2.trim.fastq.gz \\
${sampleid}_1.fastq.gz \\
${sampleid}_2.fastq.gz > ${sampleid}.cutadapt.log
"""
}
// RUN FASTQC ON CUTADAPT TRIMMED FASTQ FILES
process trim_fastqc {
tag "$sampleid"
label 'mediumcpu'
publishDir "${params.outdir}/qc/fastqc/trim", mode: 'copy',
saveAs: {filename -> filename.endsWith(".zip") ? "zip/$filename" : "$filename"}
input:
set val(sampleid), file(fastqs) from cutadapt_fastqc_ch
output:
set val(sampleid), file("*.{zip,html}") into trim_fastqc_ch
script:
"""
fastqc --outdir ./ --threads ${task.cpus} ${fastqs[0]}
fastqc --outdir ./ --threads ${task.cpus} ${fastqs[1]}
"""
}
///////////////////////////////////////////////////////////////////////////////
///////////////////////////////////////////////////////////////////////////////
/* -- -- */
/* -- ALIGN -- */
/* -- -- */
///////////////////////////////////////////////////////////////////////////////
///////////////////////////////////////////////////////////////////////////////
// RUN BWA ALN ON CUTADAPT TRIMMED FASTQ FILE FOR READ 1
process bwa_aln_r1 {
tag "$sampleid"
label 'highcpu'
publishDir "${params.outdir}/align", mode: 'copy',
saveAs: {filename ->
if (filename.endsWith(".sysout")) "sysout/$filename"
else null
}
input:
set val(sampleid), file(fastqs) from cutadapt_bwa_aln_r1_ch
file index from bwa_index_bwa_aln_r1_ch.collect()
output:
set val(sampleid), file("*.sai") into bwa_aln_r1_ch
set val(sampleid), file("*.sysout") into bwa_aln_r1_sysout_ch
script:
"""
bwa aln -t ${task.cpus} ${params.bwa_index} ${fastqs[0]} > ${sampleid}_1.sai 2> ${sampleid}_bwa_aln_r1.sysout
"""
}
// RUN BWA ALN ON CUTADAPT TRIMMED FASTQ FILE FOR READ 2
process bwa_aln_r2 {
tag "$sampleid"
label 'highcpu'
publishDir "${params.outdir}/align", mode: 'copy',
saveAs: {filename ->
if (filename.endsWith(".sysout")) "sysout/$filename"
else null
}
input:
set val(sampleid), file(fastqs) from cutadapt_bwa_aln_r2_ch
file index from bwa_index_bwa_aln_r2_ch.collect()
output:
set val(sampleid), file("*.sai") into bwa_aln_r2_ch
set val(sampleid), file("*.sysout") into bwa_aln_r2_sysout_ch
script:
"""
bwa aln -t ${task.cpus} ${params.bwa_index} ${fastqs[1]} > ${sampleid}_2.sai 2> ${sampleid}_bwa_aln_r2.sysout
"""
}
// RUN BWA SAMPE FOR SAI TO SAM CONVERSION
process bwa_sampe {
tag "$sampleid"
label "sampe"
publishDir "${params.outdir}/align", mode: 'copy',
saveAs: {filename ->
if (filename.endsWith(".sysout")) "sysout/$filename"
else null
}
input:
set val(sampleid), file(fastqs), file(sai1), file(sai2) from cutadapt_bwa_sai_to_sam_ch.join(bwa_aln_r1_ch, by: [0]).join(bwa_aln_r2_ch, by: [0])
file index from bwa_index_bwa_sampe_ch.collect()
output:
set val(sampleid), file("*.sam") into bwa_sampe_ch
set val(sampleid), file("*.sysout") into bwa_sampe_sysout_ch
script:
rg="\'@RG\\tID:${sampleid}\\tSM:${sampleid.toString().subSequence(0, sampleid.length() - 3)}\\tPL:illumina\\tLB:1\\tPU:1\'"
"""
bwa sampe -r $rg ${params.bwa_index} ${sai1} ${sai2} ${fastqs} > ${sampleid}.sam 2> ${sampleid}_bwa_sampe.sysout
"""
}
// CONVERT SAM TO COORDINATE SORTED BAM
process bwa_bam {
tag "$sampleid"
label 'lowcpu'
input:
set val(sampleid), file(sam) from bwa_sampe_ch
output:
set val(sampleid), file("*.sorted.{bam,bam.bai}") into bwa_bam_ch
set val(sampleid), file("*.flagstat") into bwa_bam_flagstat_ch
script:
out_prefix="${sampleid}"
"""
samtools view -b -h -O BAM -@ ${task.cpus} -o ${out_prefix}.bam ${sam}
samtools sort -@ ${task.cpus} -o ${out_prefix}.sorted.bam -T ${out_prefix} ${out_prefix}.bam
samtools index ${out_prefix}.sorted.bam
samtools flagstat ${out_prefix}.sorted.bam > ${out_prefix}.sorted.bam.flagstat
"""
}
///////////////////////////////////////////////////////////////////////////////
///////////////////////////////////////////////////////////////////////////////
/* -- -- */
/* -- BAM POST-PROCESSING -- */
/* -- -- */
///////////////////////////////////////////////////////////////////////////////
///////////////////////////////////////////////////////////////////////////////
// RUN PICARD MARK DUPLICATES ON COORDINATE SORTED BAM
process markdup {
tag "$sampleid"
label 'lowcpu'
publishDir "${params.outdir}/align", mode: 'copy',
saveAs: {filename ->
if (filename.endsWith(".flagstat")) "flagstat/$filename"
else if (filename.endsWith(".idxstats")) "idxstats/$filename"
else if (filename.endsWith(".sysout")) "sysout/$filename"
else if (filename.endsWith(".metrics.txt")) "picard_metrics/$filename"
else null
}
input:
set val(sampleid), file(bam) from bwa_bam_ch
output:
set val(sampleid), file("*.{bam,bam.bai}") into markdup_filter_bam_ch, markdup_collectmetrics_in_ch
set val(sampleid), file("*.flagstat") into markdup_flagstat_ch
set val(sampleid), file("*.idxstats") into markdup_idxstats_ch
set val(sampleid), file("*.metrics.txt") into markdup_metrics_ch
set val(sampleid), file("*.sysout") into markdup_sysout_ch
script:
out_prefix="${sampleid}.mkD"
"""
picard -Xmx${task.memory.toString().split(" ")[0]}g \\
MarkDuplicates \\
VALIDATION_STRINGENCY=LENIENT \\
REMOVE_DUPLICATES=false \\
ASSUME_SORTED=true \\
TMP_DIR=tmp \\
INPUT=${bam[0]} \\
OUTPUT=${out_prefix}.sorted.bam \\
METRICS_FILE=${out_prefix}.MarkDuplicates.metrics.txt \\
>> ${out_prefix}.MarkDuplicates.sysout 2>&1
samtools index ${out_prefix}.sorted.bam
samtools flagstat ${out_prefix}.sorted.bam > ${out_prefix}.sorted.bam.flagstat
samtools idxstats ${out_prefix}.sorted.bam > ${out_prefix}.sorted.bam.idxstats
"""
}
// RUN PICARD COLLECTMULTIPLE METRICS ON COORDINATE SORTED BAM
process markdup_collectmetrics {
tag "$sampleid"
label 'lowcpu'
publishDir "${params.outdir}/align", mode: 'copy',
saveAs: {filename ->
if (filename.endsWith("_metrics")) "picard_metrics/$filename"
else if (filename.endsWith(".pdf")) "picard_metrics/pdf/$filename"
else if (filename.endsWith(".sysout")) "sysout/$filename"
}
input:
set val(sampleid), file(bam) from markdup_collectmetrics_in_ch
file fasta from fasta_markdup_collectmetrics_ch.collect()
output:
set val(sampleid), file("*{_metrics,.pdf}") into markdup_collectmetrics_ch
set val(sampleid), file("*.sysout") into markdup_collectmetrics_sysout_ch
script:
out_prefix="${sampleid}.mkD"
"""
picard -Xmx${task.memory.toString().split(" ")[0]}g \\
CollectMultipleMetrics \\
VALIDATION_STRINGENCY=LENIENT \\
TMP_DIR=tmp \\
INPUT=${bam[0]} \\
OUTPUT=${out_prefix}.CollectMultipleMetrics \\
REFERENCE_SEQUENCE=${fasta} \\
>> ${out_prefix}.CollectMultipleMetrics.sysout 2>&1
"""
}
// FILTER BAM FILE TO KEEP (1) UNIQUELY MAPPED, (2) PRIMARY ALIGNMENT, (3) NON-DUPLICATES, (4) NON-MITOCHONDRIAL (5) IN FR ORIENTATION ON SAME CHROMOSOME
// (6) NON-SOFTCLIPPED (bamtools), (7) INSERT SIZE < 2KB (bamtools), (8) MISMATCH <= 3 (bamtools)
process filter_bam {
tag "$sampleid"
label 'lowcpu'
input:
set val(sampleid), file(bam) from markdup_filter_bam_ch
file include_bed from prep_genome_bed_filter_bam_ch.collect()
output:
set val(sampleid), file("*.flT.bam") into filter_bam_ch
set val(sampleid), file("*.sorted.{bam,bam.bai}") into filter_bam_sort_ch
set val(sampleid), file("*.flagstat") into filter_bam_flagstat_ch
script:
// 0x0001 = read paired
// 0x0004 = read unmapped
// 0x0008 = mate unmapped
// 0x0100 = not primary alignment
// 0x0400 = read is PCR or optical duplicate
out_prefix="${sampleid}.flT"
"""
samtools view \\
-f 0x001 \\
-F 0x004 \\
-F 0x0008 \\
-F 0x0100 \\
-F 0x0400 \\
-q 1 \\
-L ${include_bed} \\
-b ${bam[0]} \\
| bamtools filter \\
-out ${out_prefix}.sorted.bam \\
-script ${bamtools_filter_pe_config}
samtools index ${out_prefix}.sorted.bam
samtools flagstat ${out_prefix}.sorted.bam > ${out_prefix}.sorted.bam.flagstat
samtools sort -n -@ ${task.cpus} -o ${out_prefix}.bam -T ${out_prefix} ${out_prefix}.sorted.bam
"""
}
// FILTER BAM FILE TO REMOVE READS WHERE ONE MATE HAS BEEN PHYSICALLY FILTERED OUT OF BAM FILE FROM PREVIOUS STEP.
process rm_orphan {
tag "$sampleid"
input:
set val(sampleid), file(bam) from filter_bam_ch
output:
set val(sampleid), file("*.bam") into rm_orphan_ch
script:
out_prefix="${sampleid}.clN"
"""
python bampe_rm_orphan.py ${bam} ${out_prefix}.bam --only_prop_pair
"""
}
// SORT ORPHAN BAM FILE BY COORDINATE
process rm_orphan_sort_bam {
tag "$sampleid"
label 'lowcpu'
publishDir "${params.outdir}/align", mode: 'copy',
saveAs: {filename ->
if (filename.endsWith(".flagstat")) "flagstat/$filename"
else if (filename.endsWith(".idxstats")) "idxstats/$filename"
else if (filename.endsWith(".bam")) "$filename"
else if (filename.endsWith(".bai")) "$filename"
}
input:
set val(sampleid), file(filter_bam), file(orphan_bam) from filter_bam_sort_ch.join(rm_orphan_ch, by: [0])
output:
set val(sampleid), file("*.{bam,bam.bai,flagstat}") into rm_orphan_sort_bam_replicate_ch,
rm_orphan_sort_bam_sample_ch,
rm_orphan_sort_bam_replicate_rmdup_ch,
rm_orphan_sort_bam_sample_rmdup_ch
set val(sampleid), file("*.idxstats") into rm_orphan_sort_bam_idxstats_ch
script:
out_prefix="${sampleid}.clN"
"""
samtools sort -@ ${task.cpus} -o ${out_prefix}.sorted.bam -T ${out_prefix} ${out_prefix}.bam
samtools index ${out_prefix}.sorted.bam
samtools flagstat ${out_prefix}.sorted.bam > ${out_prefix}.sorted.bam.flagstat
samtools idxstats ${out_prefix}.sorted.bam > ${out_prefix}.sorted.bam.idxstats
"""
}
///////////////////////////////////////////////////////////////////////////////
///////////////////////////////////////////////////////////////////////////////
/* -- -- */
/* -- MERGE REPLICATE BAM -- */
/* -- -- */
///////////////////////////////////////////////////////////////////////////////
///////////////////////////////////////////////////////////////////////////////
// CREATE CHANNEL TO MERGE AT REPLICATE LEVEL
rm_orphan_sort_bam_replicate_ch.map { it -> [ it[0].toString().subSequence(0, it[0].length() - 3), it[1] ] }
.groupTuple(by: [0])
.map { it -> [ it[0], it[1].flatten() ] }
.into { rm_orphan_sort_bam_replicate_merge_ch;
rm_orphan_sort_bam_replicate_markdup_ch;
rm_orphan_sort_bam_replicate_rmdup_ch }
// MERGE FILTERED BAM FILES AT REPLICATE LEVEL USING PICARD MERGESAMFILES
process merge_replicate {
tag "$sampleid"
label 'lowcpu'
publishDir "${params.outdir}/align/replicateLevel", mode: 'copy',
saveAs: {filename ->
if (filename.endsWith(".sysout")) "sysout/$filename"
else null
}
input:
set val(sampleid), file(bams) from rm_orphan_sort_bam_replicate_merge_ch
output:
set val(sampleid), file("*.{bam,bam.bai}") into merge_replicate_ch
set val(sampleid), file("*.flagstat") into merge_replicate_flagstat_ch
set val(sampleid), file("*.sysout") into merge_replicate_sysout_ch
script:
out_prefix="${sampleid}.RpL"
bam_files = bams.findAll { it.toString().endsWith('.bam') }.sort()
flagstat_files = bams.findAll { it.toString().endsWith('.flagstat') }.sort()
if (bam_files.size() > 1) {
"""
picard -Xmx${task.memory.toString().split(" ")[0]}g \\
MergeSamFiles \\
VALIDATION_STRINGENCY=LENIENT \\
SORT_ORDER=coordinate \\
TMP_DIR=tmp \\
${'INPUT='+bam_files.join(' INPUT=')} \\
OUTPUT=${out_prefix}.sorted.bam \\
>> ${out_prefix}.MergeSamFiles.sysout 2>&1
samtools index ${out_prefix}.sorted.bam
samtools flagstat ${out_prefix}.sorted.bam > ${out_prefix}.sorted.bam.flagstat
"""
} else {
"""
touch ${out_prefix}.sorted.bam
touch ${out_prefix}.sorted.bam.bai
touch ${out_prefix}.MergeSamFiles.sysout
cp ${flagstat_files[0]} ${out_prefix}.sorted.bam.flagstat
"""
}
}
// RUN PICARD MARK DUPLICATES
process merge_replicate_markdup {
tag "$sampleid"
label 'lowcpu'
publishDir "${params.outdir}/align/replicateLevel", mode: 'copy',
saveAs: {filename ->
if (filename.endsWith(".sysout")) "sysout/$filename"
else if (filename.endsWith(".metrics.txt")) "picard_metrics/$filename"
else null
}
input:
set val(sampleid), file(orphan_bams), file(merged_bam) from rm_orphan_sort_bam_replicate_markdup_ch.join(merge_replicate_ch, by: [0])
output:
set val(sampleid), file("*.{bam,bam.bai}") into merge_replicate_markdup_ch
set val(sampleid), file("*.flagstat") into merge_replicate_markdup_flagstat_ch
set val(sampleid), file("*.metrics.txt") into merge_replicate_markdup_metrics_ch
set val(sampleid), file("*.sysout") into merge_replicate_markdup_sysout_ch
script:
out_prefix="${sampleid}.RpL.mkD"
bam_files = orphan_bams.findAll { it.toString().endsWith('.bam') }.sort()
flagstat_files = orphan_bams.findAll { it.toString().endsWith('.flagstat') }.sort()
if (bam_files.size() > 1) {
"""
picard -Xmx${task.memory.toString().split(" ")[0]}g \\
MarkDuplicates \\
VALIDATION_STRINGENCY=LENIENT \\
REMOVE_DUPLICATES=false \\
ASSUME_SORTED=true \\
TMP_DIR=tmp \\
INPUT=${merged_bam[0]} \\
OUTPUT=${out_prefix}.sorted.bam \\
METRICS_FILE=${out_prefix}.MarkDuplicates.metrics.txt \\
>> ${out_prefix}.MarkDuplicates.sysout 2>&1
samtools index ${out_prefix}.sorted.bam
samtools flagstat ${out_prefix}.sorted.bam > ${out_prefix}.sorted.bam.flagstat
"""
} else {
"""
touch ${out_prefix}.sorted.bam
touch ${out_prefix}.sorted.bam.bai
touch ${out_prefix}.MarkDuplicates.sysout
touch ${out_prefix}.MarkDuplicates.metrics.txt
cp ${flagstat_files[0]} ${out_prefix}.sorted.bam.flagstat
"""
}
}
// REMOVE DUPLICATES USING SAMTOOLS
process merge_replicate_rmdup {
tag "$sampleid"
publishDir "${params.outdir}/align/replicateLevel", mode: 'copy',
saveAs: {filename ->
if (filename.endsWith(".flagstat")) "flagstat/$filename"
else if (filename.endsWith(".bam")) "$filename"
else if (filename.endsWith(".bai")) "$filename"
}
input:
set val(sampleid), file(orphan_bams), file(markdup_bam) from rm_orphan_sort_bam_replicate_rmdup_ch.join(merge_replicate_markdup_ch, by: [0])
output:
set val(sampleid), file("*.{bam,bam.bai}") into merge_replicate_rmdup_name_bam_ch,
merge_replicate_rmdup_bedgraph_ch,
merge_replicate_rmdup_macs2_ch,
merge_replicate_rmdup_macs2_frip_ch
set val(sampleid), file("*.flagstat") into merge_replicate_rmdup_flagstat_ch,
merge_replicate_rmdup_flagstat_bedgraph_ch,
merge_replicate_rmdup_flagstat_macs2_frip_ch
script:
out_prefix="${sampleid}.RpL.rmD"
bam_files = orphan_bams.findAll { it.toString().endsWith('.bam') }.sort()
if (bam_files.size() > 1) {
"""
samtools view -bF 0x400 ${markdup_bam[0]} > ${out_prefix}.sorted.bam
samtools index ${out_prefix}.sorted.bam
samtools flagstat ${out_prefix}.sorted.bam > ${out_prefix}.sorted.bam.flagstat
"""
} else {
"""
cp ${bam_files[0]} ${out_prefix}.sorted.bam
samtools index ${out_prefix}.sorted.bam
samtools flagstat ${out_prefix}.sorted.bam > ${out_prefix}.sorted.bam.flagstat
"""
}
}
// SORT BAM FILE BY NAME
process merge_replicate_name_bam {
tag "$sampleid"
label 'lowcpu'
input:
set val(sampleid), file(bam) from merge_replicate_rmdup_name_bam_ch
output:
set val(sampleid), file("*.bam") into merge_replicate_name_bam_merge_replicate_featurecounts_ch,
merge_replicate_name_bam_merge_sample_featurecounts_ch
script:
out_prefix="${sampleid}.RpL.rmD"
"""
samtools sort -n -@ ${task.cpus} -o ${out_prefix}.bam -T ${out_prefix} ${out_prefix}.sorted.bam
"""
}
///////////////////////////////////////////////////////////////////////////////
///////////////////////////////////////////////////////////////////////////////
/* -- -- */
/* -- MERGE REPLICATE BAM POST-ANALYSIS -- */
/* -- -- */
///////////////////////////////////////////////////////////////////////////////
///////////////////////////////////////////////////////////////////////////////
////////////////////////////////////////////////////
/* -- COVERAGE TRACKS -- */
////////////////////////////////////////////////////
// CREATE NORMALISED BEDGRAPH FILE USING BEDTOOLS GENOMECOVERAGEBED
// CALCULATE SCALE-FACTOR FROM FLAGSTAT FILE
process merge_replicate_bedgraph {
tag "$sampleid"
publishDir "${params.outdir}/align/replicateLevel/bigwig", mode: 'copy',
saveAs: {filename ->
if (filename.endsWith(".txt")) "scale_factor/$filename"
else null
}
input:
set val(sampleid), file(bam), file(flagstat) from merge_replicate_rmdup_bedgraph_ch.join(merge_replicate_rmdup_flagstat_bedgraph_ch, by: [0])
file chrom_sizes from prep_genome_sizes_replicate_bedgraph_ch.collect()
output:
set val(sampleid), file("*.bg") into merge_replicate_bedgraph_ch
set val(sampleid), file("*.txt") into merge_replicate_bedgraph_scale_ch
script:
out_prefix="${sampleid}.RpL.rmD"
"""
SCALE_FACTOR=\$(grep 'read1' ${flagstat} | awk '{print 1000000/\$1}')
echo \$SCALE_FACTOR > ${out_prefix}.scale_factor.txt
genomeCoverageBed -ibam ${bam[0]} -bg -trackline -scale \$SCALE_FACTOR -pc -g ${chrom_sizes} > ${out_prefix}.bg
"""
}
// CONVERT BEDGRAPH TO BIGWIG USING KENTOOLS
process merge_replicate_bigwig {
tag "$sampleid"
label 'bigwig'
publishDir "${params.outdir}/align/replicateLevel/bigwig", mode: 'copy'
input:
set val(sampleid), file(bedgraph) from merge_replicate_bedgraph_ch
file chrom_sizes from prep_genome_sizes_replicate_bigwig_ch.collect()
output:
set val(sampleid), file("*.bigWig") into merge_replicate_bigwig_ch
script:
out_prefix="${sampleid}.RpL.rmD"
"""
wigToBigWig -clip ${bedgraph} ${chrom_sizes} ${out_prefix}.bigWig
"""
}
////////////////////////////////////////////////////
/* -- MACS2 BROAD -- */
////////////////////////////////////////////////////
// CALL BROAD PEAKS USING MACS2
process merge_replicate_macs2 {
tag "$sampleid"
publishDir "${params.outdir}/align/replicateLevel/macs2", mode: 'copy'
input:
set val(sampleid), file(bam) from merge_replicate_rmdup_macs2_ch
output:
set val(sampleid), file("*/*.broadPeak") into merge_replicate_macs2_homer_in_ch,
merge_replicate_macs2_frip_in_ch,
merge_replicate_macs2_merge_peaks_in_ch,
merge_replicate_macs2_igv_in_ch
set val(sampleid), file("*/*.{gappedPeak,xls}") into merge_replicate_macs2_output_ch
set val(sampleid), file("*/*/*.sysout") into merge_replicate_macs2_sysout_ch