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#!/usr/bin/env nextflow
/*
========================================================================================
C H I P - S E Q / A T A C - S E Q B E S T P R A C T I C E
========================================================================================
ChIP-seq/ATAC-seq Best Practice Analysis Pipeline. Started May 2016.
#### Homepage / Documentation
https://github.com/nf-core/chipseq
@#### Authors
Chuan Wang <[email protected]>
Phil Ewels <[email protected]>
Alex Peltzer <[email protected]>
----------------------------------------------------------------------------------------
----------------------------------------------------------------------------------------
Pipeline overview:
- 1: FastQC for raw sequencing reads quality control
- 2: Trim Galore! for adapter trimming
- 3.1: BWA alignment against reference genome
- 3.2: Post-alignment processing and format conversion
- 3.3: Statistics about mapped reads
- 4: Picard for duplicate read identification
- 5: Statistics about read counts
- 6.1: Phantompeakqualtools for normalized strand cross-correlation (NSC) and relative strand cross-correlation (RSC)
- 6.2: Summarize NSC and RSC
- 7: deepTools for fingerprint, coverage bigwig, correlation plots of reads over genome-wide bins, and distribution of reads around gene bodies
- 8.1: MACS for peak calling
- 8.2: Saturation analysis using MACS when specified
- 9: Post peak calling processing: blacklist filtering and annotation
- 10: MultiQC
- 11: Output Description HTML
----------------------------------------------------------------------------------------
*/
def helpMessage() {
log.info """
=======================================================
,--./,-.
___ __ __ __ ___ /,-._.--~\'
|\\ | |__ __ / ` / \\ |__) |__ } {
| \\| | \\__, \\__/ | \\ |___ \\`-._,-`-,
`._,._,\'
nf-core/chipseq : ChIP-Seq/ATAC-Seq Best Practice v${params.pipelineVersion}
=======================================================
Usage:
The typical command for running the pipeline is as follows:
nextflow run nf-core/chipseq --reads '*_R{1,2}.fastq.gz' --genome GRCh37 --macsconfig 'macssetup.config' -profile uppmax
Mandatory arguments:
--reads Path to input data (must be surrounded with quotes).
--genome Name of iGenomes reference
--macsconfig Configuration file for peaking calling using MACS. Format: ChIPSampleID,CtrlSampleID,AnalysisID
-profile Hardware config to use. uppmax / uppmax_modules / docker / aws
Options:
--singleEnd Specifies that the input is single end reads
--allow_multi_align Secondary alignments and unmapped reads are also reported in addition to primary alignments
--saturation Run saturation analysis by peak calling with subsets of reads
--macsgsize Effective genome size for the MACS --gsize option. Should be in the format "2.1e9"
--broad Run MACS with the --broad flag
--blacklist_filtering Filter ENCODE blacklisted regions from ChIP-seq peaks. It only works when --genome is set as GRCh37 or GRCm38
--ATACseq Apply special analysis setup for ATAC-seq dataset: strand shift will be performed, and --broad flag will be used when running MACS
Presets:
--extendReadsLen [int] Number of base pairs to extend the reads for the deepTools analysis. Default: 100
References
--fasta Path to Fasta reference
--bwa_index Path to BWA index
--largeRef Build BWA Index for large reference genome (>2Gb)
--gtf Path to GTF file (Ensembl format)
--bed Path to BED file (Ensembl format)
--skipDupRemoval Skip duplication removal by picard
--blacklist Path to blacklist regions (.BED format), used for filtering out called peaks. Note that --blacklist_filtering is required
--saveReference Save the generated reference files in the Results directory.
--saveAlignedIntermediates Save the intermediate BAM files from the Alignment step - not done by default
Trimming options
--notrim Specifying --notrim will skip the adapter trimming step.
--saveTrimmed Save the trimmed Fastq files in the the Results directory.
--clip_r1 [int] Instructs Trim Galore to remove bp from the 5' end of read 1 (or single-end reads)
--clip_r2 [int] Instructs Trim Galore to remove bp from the 5' end of read 2 (paired-end reads only)
--three_prime_clip_r1 [int] Instructs Trim Galore to remove bp from the 3' end of read 1 AFTER adapter/quality trimming has been performed
--three_prime_clip_r2 [int] Instructs Trim Galore to re move bp from the 3' end of read 2 AFTER adapter/quality trimming has been performed
Other options:
--outdir The output directory where the results will be saved
--email Set this parameter to your e-mail address to get a summary e-mail with details of the run sent to you when the workflow exits
--rlocation Location to save R-libraries used in the pipeline. Default value is ~/R/nxtflow_libs/
--project Project ID when running pipeline with slurm on UPPMAX clusters
--clusterOptions Extra SLURM options, used in conjunction with Uppmax.config
--seqCenter Text about sequencing center which will be added in the header of output bam files
-name Name for the pipeline run. If not specified, Nextflow will automatically generate a random mnemonic
""".stripIndent()
}
/*
* SET UP CONFIGURATION VARIABLES
*/
// Show help emssage
params.help = false
if (params.help){
helpMessage()
exit 0
}
// Configurable variables
params.name = false
params.project = false
params.genome = false
params.genomes = false
params.fasta = params.genome ? params.genomes[ params.genome ].fasta ?: false : false
params.bwa_index = params.genome ? params.genomes[ params.genome ].bwa ?: false : false
params.gtf = params.genome ? params.genomes[ params.genome ].gtf ?: false : false
params.bed = params.genome ? params.genomes[ params.genome ].bed ?: false : false
params.blacklist = params.genome ? params.genomes[ params.genome ].blacklist ?: false : false
params.macsgsize = params.genome ? params.genomes[ params.genome ].macsgsize ?: false : false
phantompeakqualtools_mqc_header = file("$baseDir/assets/phantompeakqualtools_mqc_header")
// R library locations
params.rlocation = false
if (params.rlocation){
nxtflow_libs = file(params.rlocation)
nxtflow_libs.mkdirs()
}
multiqc_config = file(params.multiqc_config)
output_docs = file("$baseDir/docs/output.md")
// Custom trimming options
params.clip_r1 = 0
params.clip_r2 = 0
params.three_prime_clip_r1 = 0
params.three_prime_clip_r2 = 0
// Validate inputs
macsconfig = file(params.macsconfig)
if( !macsconfig.exists() ) exit 1, "Missing MACS config: '$macsconfig'. Specify path with --macsconfig"
if( params.bwa_index ){
bwa_index = Channel
.fromPath(params.bwa_index)
.ifEmpty { exit 1, "BWA index not found: ${params.bwa_index}" }
bwa_index_genomesizetable = Channel
.fromPath(params.bwa_index)
} else if ( params.fasta ){
fasta = file(params.fasta)
if( !fasta.exists() ) exit 1, "Fasta file not found: ${params.fasta}"
} else {
exit 1, "No reference genome specified!"
}
gtf = false
if( params.gtf ){
gtf = file(params.gtf)
if( !gtf.exists() ) exit 1, "GTF file not found: ${params.gtf}."
}
bed = false
if( params.bed ){
bed = file(params.bed)
if( !bed.exists() ) exit 1, "BED file not found: ${params.bed}."
}
if ( params.blacklist_filtering ){
blacklist = file(params.blacklist)
if( !blacklist.exists() ) exit 1, "Blacklist file not found: ${params.blacklist}"
}
if( workflow.profile == 'standard' && !params.project ) exit 1, "No UPPMAX project ID found! Use --project"
// Has the run name been specified by the user?
// this has the bonus effect of catching both -name and --name
custom_runName = params.name
if( !(workflow.runName ==~ /[a-z]+_[a-z]+/) ){
custom_runName = workflow.runName
}
if(params.readPaths){
if(params.singleEnd){
Channel
.from(params.readPaths)
.map { row -> [ row[0], [file(row[1][0])]] }
.ifEmpty { exit 1, "params.readPaths was empty - no input files supplied" }
.into { raw_reads_configvali; raw_reads_fastqc; raw_reads_trimgalore }
} else {
Channel
.from(params.readPaths)
.map { row -> [ row[0], [file(row[1][0]), file(row[1][1])]] }
.ifEmpty { exit 1, "params.readPaths was empty - no input files supplied" }
.into { raw_reads_configvali; raw_reads_fastqc; raw_reads_trimgalore }
}
} else {
Channel
.fromFilePairs( params.reads, size: params.singleEnd ? 1 : 2 )
.ifEmpty { exit 1, "Cannot find any reads matching: ${params.reads}\nNB: Path needs to be enclosed in quotes!\nIf this is single-end data, please specify --singleEnd on the command line." }
.into { raw_reads_configvali; raw_reads_fastqc; raw_reads_trimgalore }
}
/*
* Create a channel for macs config file
*/
Channel
.from(macsconfig.readLines())
.map { line ->
list = line.split(',')
chip_sample_id = list[0]
ctrl_sample_id = list[1]
analysis_id = list[2]
[ chip_sample_id, ctrl_sample_id, analysis_id ]
}
.into { vali_para; macs_para; saturation_para }
// Validate all samples in macs config file
def config_samples = []
for (line in vali_para){
if (line.getClass().toString() != "class groovyx.gpars.dataflow.operator.PoisonPill") {
config_samples.add(line[0])
config_samples.add(line[1])
}
}
config_samples.removeAll{ it == '' }
config_samples.unique(false)
def fastq_samples = []
for (sample in raw_reads_configvali){
if (sample.getClass().toString() != "class groovyx.gpars.dataflow.operator.PoisonPill") {
fastq_samples.add(sample[0].toString() - ~/(.R)?(_R)?(.R1)?(_1)?(_R1)?(_trimmed)?(_val_1)?(\.fq)?(\.fastq)?(\.gz)?$/)
}
}
def missing_samples = config_samples - config_samples.intersect(fastq_samples)
if(!missing_samples.isEmpty()){
exit 1, "No FastQ file found for sample in MACS config: ${missing_samples}"
}
def dropped_samples = fastq_samples - fastq_samples.intersect(config_samples)
if(!dropped_samples.isEmpty()){
exit 1, "Sample ${dropped_samples} not included in MACS config"
}
log.info """=======================================================
,--./,-.
___ __ __ __ ___ /,-._.--~\'
|\\ | |__ __ / ` / \\ |__) |__ } {
| \\| | \\__, \\__/ | \\ |___ \\`-._,-`-,
`._,._,\'
nf-core/chipseq : ChIP-Seq Best Practice v${params.pipelineVersion}
======================================================="""
def summary = [:]
summary['Run Name'] = custom_runName ?: workflow.runName
summary['ATAC-seq'] = params.ATACseq
summary['Reads'] = params.reads
summary['Data Type'] = params.singleEnd ? 'Single-End' : 'Paired-End'
summary['Genome'] = params.genome
if(params.bwa_index) summary['BWA Index'] = params.bwa_index
else if(params.fasta) summary['Fasta Ref'] = params.fasta
if(params.gtf) summary['GTF File'] = params.gtf
if(params.bed) summary['BED File'] = params.bed
if(params.largeRef) summary['Build BWA Index for Large Reference'] = params.largeRef
summary['Multiple alignments'] = params.allow_multi_align
summary['Skip Duplication Removal'] = params.skipDupRemoval
summary['MACS Config'] = params.macsconfig
summary['MACS gsize'] = params.macsgsize
summary['Saturation analysis'] = params.saturation
summary['MACS broad peaks'] = params.broad
summary['Blacklist filtering'] = params.blacklist_filtering
if(params.blacklist_filtering) summary['Blacklist BED'] = params.blacklist
summary['Extend Reads'] = "$params.extendReadsLen bp"
if(workflow.container) summary['Container'] = workflow.container
if(workflow.revision) summary['Pipeline Release'] = workflow.revision
summary['Current home'] = "$HOME"
summary['Current user'] = "$USER"
summary['Current path'] = "$PWD"
summary['Working dir'] = workflow.workDir
summary['Output dir'] = params.outdir
summary['R libraries'] = params.rlocation
summary['Script dir'] = workflow.projectDir
summary['Save Reference'] = params.saveReference
summary['Save Trimmed'] = params.saveTrimmed
summary['Save Intermeds'] = params.saveAlignedIntermediates
if( params.notrim ){
summary['Trimming Step'] = 'Skipped'
} else {
summary['Trim R1'] = params.clip_r1
summary['Trim R2'] = params.clip_r2
summary["Trim 3' R1"] = params.three_prime_clip_r1
summary["Trim 3' R2"] = params.three_prime_clip_r2
}
summary['Config Profile'] = workflow.profile
if(params.seqCenter) summary['Seq Center'] = params.seqCenter
if(params.project) summary['UPPMAX Project'] = params.project
if(params.email) summary['E-mail Address'] = params.email
if(workflow.commitId) summary['Pipeline Commit']= workflow.commitId
log.info summary.collect { k,v -> "${k.padRight(21)}: $v" }.join("\n")
log.info "===================================="
// Show a big error message if we're running on the base config and an uppmax cluster
if( workflow.profile == 'standard'){
if ( "hostname".execute().text.contains('.uppmax.uu.se') ) {
log.error "====================================================\n" +
" WARNING! You are running with the default 'standard'\n" +
" pipeline config profile, which runs on the head node\n" +
" and assumes all software is on the PATH.\n" +
" ALL JOBS ARE RUNNING LOCALLY and stuff will probably break.\n" +
" Please use `-profile uppmax` to run on UPPMAX clusters.\n" +
"============================================================"
}
}
/*
* PREPROCESSING - Build BWA index
*/
if(!params.bwa_index && fasta){
process makeBWAindex {
tag fasta
publishDir path: { params.saveReference ? "${params.outdir}/reference_genome" : params.outdir },
saveAs: { params.saveReference ? it : null }, mode: 'copy'
input:
file fasta from fasta
output:
file "BWAIndex" into bwa_index_genomesizetable, bwa_index
script:
BWAIndexOption = params.largeRef ? "bwtsw" : 'is'
"""
bwa index -a $BWAIndexOption $fasta
mkdir BWAIndex && mv ${fasta}* BWAIndex
"""
}
}
/*
* PREPROCESSING - Build BED file
*/
if(!params.bed){
process makeBED {
tag "$gtf"
publishDir path: { params.saveReference ? "${params.outdir}/reference_genome" : params.outdir },
saveAs: { params.saveReference ? it : null }, mode: 'copy'
input:
file gtf from gtf
output:
file "${gtf.baseName}.bed" into bed
script: // This script is bundled with the pipeline, in nfcore/chipseq/bin/
"""
gtf2bed $gtf > ${gtf.baseName}.bed
"""
}
}
/*
* PREPROCESSING - Prepare genome size table for ATAC-seq
*/
if(params.ATACseq){
process makeGenomeSizeTable {
publishDir path: { params.saveReference ? "${params.outdir}/reference_genome" : params.outdir },
saveAs: { params.saveReference ? it : null }, mode: 'copy'
input:
file index from bwa_index_genomesizetable.collect()
output:
file "*.txt" into genomeSizeTable
script:
"""
ln -s ${index}/genome.fa genome.fa
samtools faidx genome.fa > genome.fa.fai
cut -f1,2 genome.fa.fai > genomeSizeTable.txt
"""
}
}
/*
* STEP 1 - FastQC
*/
process fastqc {
tag "$name"
publishDir "${params.outdir}/fastqc", mode: 'copy',
saveAs: {filename -> filename.indexOf(".zip") > 0 ? "zips/$filename" : "$filename"}
input:
set val(name), file(reads) from raw_reads_fastqc
output:
file '*_fastqc.{zip,html}' into fastqc_results
file '.command.out' into fastqc_stdout
script:
"""
fastqc -q $reads
"""
}
/*
* STEP 2 - Trim Galore!
*/
if(params.notrim){
trimmed_reads = read_files_trimming
trimgalore_results = []
trimgalore_fastqc_reports = []
} else {
process trim_galore {
tag "$name"
publishDir "${params.outdir}/trim_galore", mode: 'copy',
saveAs: {filename ->
if (filename.indexOf("_fastqc") > 0) "FastQC/$filename"
else if (filename.indexOf("trimming_report.txt") > 0) "logs/$filename"
else params.saveTrimmed ? filename : null
}
input:
set val(name), file(reads) from raw_reads_trimgalore
output:
file '*.fq.gz' into trimmed_reads
file '*trimming_report.txt' into trimgalore_results
file "*_fastqc.{zip,html}" into trimgalore_fastqc_reports
script:
c_r1 = params.clip_r1 > 0 ? "--clip_r1 ${params.clip_r1}" : ''
c_r2 = params.clip_r2 > 0 ? "--clip_r2 ${params.clip_r2}" : ''
tpc_r1 = params.three_prime_clip_r1 > 0 ? "--three_prime_clip_r1 ${params.three_prime_clip_r1}" : ''
tpc_r2 = params.three_prime_clip_r2 > 0 ? "--three_prime_clip_r2 ${params.three_prime_clip_r2}" : ''
if (params.singleEnd) {
"""
trim_galore --fastqc --gzip $c_r1 $tpc_r1 $reads
"""
} else {
"""
trim_galore --paired --fastqc --gzip $c_r1 $c_r2 $tpc_r1 $tpc_r2 $reads
"""
}
}
}
/*
* STEP 3.1 - align with bwa
*/
process bwa {
tag "$prefix"
publishDir path: { params.saveAlignedIntermediates ? "${params.outdir}/bwa" : params.outdir }, mode: 'copy',
saveAs: {filename -> params.saveAlignedIntermediates ? filename : null }
input:
file reads from trimmed_reads
file index from bwa_index.collect()
output:
file '*.bam' into bwa_bam
script:
prefix = reads[0].toString() - ~/(.R1)?(_1)?(_R1)?(_trimmed)?(_val_1)?(\.fq)?(\.fastq)?(\.gz)?$/
filtering = params.allow_multi_align ? '' : "| samtools view -b -q 1 -F 4 -F 256"
seqCenter = params.seqCenter ? "-R '@RG\\tID:${prefix}\\tCN:${params.seqCenter}'" : ''
"""
bwa mem -M $seqCenter ${index}/genome.fa $reads | samtools view -bT $index - $filtering > ${prefix}.bam
"""
}
/*
* STEP 3.2 - post-alignment processing
*/
if(!params.ATACseq){
process samtools_chipseq {
tag "${bam.baseName}"
publishDir path: "${params.outdir}/bwa", mode: 'copy',
saveAs: { filename ->
if (filename.indexOf(".stats.txt") > 0) "stats/$filename"
else params.saveAlignedIntermediates ? filename : null
}
input:
file bam from bwa_bam
output:
file '*.sorted.bam' into bam_picard, bam_for_mapped
file '*.sorted.bam.bai' into bai_picard, bai_for_mapped
file '*.sorted.bed' into bed_total
file '*.stats.txt' into samtools_stats
script:
"""
samtools sort $bam -o ${bam.baseName}.sorted.bam
samtools index ${bam.baseName}.sorted.bam
bedtools bamtobed -i ${bam.baseName}.sorted.bam | sort -k 1,1 -k 2,2n -k 3,3n -k 6,6 > ${bam.baseName}.sorted.bed
samtools stats ${bam.baseName}.sorted.bam > ${bam.baseName}.stats.txt
"""
}
} else {
process samtools_atacseq {
tag "${bam.baseName}"
publishDir path: "${params.outdir}/bwa", mode: 'copy',
saveAs: { filename ->
if (filename.indexOf(".stats.txt") > 0) "stats/$filename"
else params.saveAlignedIntermediates ? filename : null
}
input:
file bam from bwa_bam
file genomeSizeTable from genomeSizeTable
output:
file '*.sorted.bam' into bam_picard, bam_for_mapped
file '*.sorted.bam.bai' into bai_picard, bai_for_mapped
file '*.sorted.bed' into bed_total
file '*.stats.txt' into samtools_stats
script:
"""
samtools sort $bam | bedtools bamtobed | awk -v OFS="\\t" '{if(\$6=="+"){\$2+=4;\$3+=4;print} else if(\$6=="-" && \$2>5){\$2-=5;\$3-=5;print} else if(\$6=="-" && \$2<=5){\$3=\$3-\$2+1;\$2=1;print}}' | sort -k 1,1 -k 2,2n -k 3,3n -k 6,6 > ${bam.baseName}.sorted.bed
bedToBam -i ${bam.baseName}.sorted.bed -g $genomeSizeTable | samtools sort > ${bam.baseName}.sorted.bam
samtools index ${bam.baseName}.sorted.bam
samtools stats ${bam.baseName}.sorted.bam > ${bam.baseName}.stats.txt
"""
}
}
/*
* STEP 3.3 - Statistics about mapped and unmapped reads against ref genome
*/
process bwa_mapped {
tag "${input_files[0].baseName}"
publishDir "${params.outdir}/bwa/mapped", mode: 'copy'
input:
file input_files from bam_for_mapped.collect()
file bai from bai_for_mapped.collect()
output:
file 'mapped_refgenome.txt' into bwa_mapped
script:
"""
for i in $input_files
do
samtools idxstats \${i} | awk -v filename="\${i}" '{mapped+=\$3; unmapped+=\$4} END {print filename,"\t",mapped,"\t",unmapped}'
done > mapped_refgenome.txt
"""
}
/*
* STEP 4 Picard
*/
if (params.skipDupRemoval) {
bam_picard.into {bam_dedup_spp; bam_dedup_deepTools; bam_dedup_macs; bam_dedup_saturation}
bai_picard.into {bai_dedup_spp; bai_dedup_deepTools; bai_dedup_macs; bai_dedup_saturation}
picard_reports = Channel.from(false)
} else {
process picard {
tag "$prefix"
publishDir "${params.outdir}/picard", mode: 'copy'
input:
file bam from bam_picard
file bai from bai_picard
output:
file '*.dedup.sorted.bam' into bam_dedup_spp, bam_dedup_deepTools, bam_dedup_macs, bam_dedup_saturation
file '*.dedup.sorted.bam.bai' into bai_dedup_spp, bai_dedup_deepTools, bai_dedup_macs, bai_dedup_saturation
file '*.dedup.sorted.bed' into bed_dedup
file '*.picardDupMetrics.txt' into picard_reports
script:
prefix = bam[0].toString() - ~/(\.sorted)?(\.bam)?$/
if( ! task.memory ){
log.warn "[Picard MarkDuplicates] Available memory not known - defaulting to 6GB ($prefix)"
avail_mem = 6000
} else {
avail_mem = task.memory.toMega()
if( avail_mem <= 0){
avail_mem = 6000
log.warn "[Picard MarkDuplicates] Available memory 0 - defaulting to 6GB ($prefix)"
} else if( avail_mem < 250){
avail_mem = 250
log.warn "[Picard MarkDuplicates] Available memory under 250MB - defaulting to 250MB ($prefix)"
}
}
"""
picard MarkDuplicates \\
INPUT=$bam \\
OUTPUT=${prefix}.dedup.bam \\
ASSUME_SORTED=true \\
REMOVE_DUPLICATES=true \\
METRICS_FILE=${prefix}.picardDupMetrics.txt \\
VALIDATION_STRINGENCY=LENIENT \\
PROGRAM_RECORD_ID='null'
samtools sort ${prefix}.dedup.bam -o ${prefix}.dedup.sorted.bam
samtools index ${prefix}.dedup.sorted.bam
bedtools bamtobed -i ${prefix}.dedup.sorted.bam | sort -k 1,1 -k 2,2n -k 3,3n -k 6,6 > ${prefix}.dedup.sorted.bed
"""
}
}
/*
* STEP 5 Read_count_statistics
*/
process countstat {
tag "${bed[0].baseName}"
publishDir "${params.outdir}/countstat", mode: 'copy'
input:
file bed from params.skipDupRemoval ? bed_total.collect() : bed_total.mix(bed_dedup).collect()
output:
file 'read_count_statistics.txt' into countstat_results
script:
"""
countstat.pl $bed
"""
}
/*
* STEP 6.1 Phantompeakqualtools
* TODO: The "run_spp.R" script is still missing here!
*/
process phantompeakqualtools {
tag "$prefix"
publishDir "${params.outdir}/phantompeakqualtools", mode: 'copy',
saveAs: {filename -> filename.indexOf(".out") > 0 ? "logs/$filename" : "$filename"}
input:
file bam from bam_dedup_spp
file bai from bai_dedup_spp
file phantompeakqualtools_mqc_header
output:
file '*.pdf' into spp_plot
file '*.spp.out' into spp_out, spp_out_mqc
file '*_mqc.csv' into spp_csv_mqc
script:
prefix = bam[0].toString() - ~/(\.dedup)?(\.sorted)?(\.bam)?$/
"""
run_spp.r -c="$bam" -savp -savd="${prefix}.spp.Rdata" -out="${prefix}.spp.out"
processSppRdata.r ${prefix}.spp.Rdata ${prefix}.spp.csv
cat $phantompeakqualtools_mqc_header ${prefix}.spp.csv > ${prefix}_mqc.csv
"""
}
/*
* STEP 6.2 Combine and calculate NSC & RSC
*/
process calculateNSCRSC {
tag "${spp_out_list[0].baseName}"
publishDir "${params.outdir}/phantompeakqualtools", mode: 'copy'
input:
file spp_out_list from spp_out.collect()
output:
file 'cross_correlation_processed.txt' into calculateNSCRSC_results
script:
"""
cat $spp_out_list > cross_correlation.txt
calculateNSCRSC.r cross_correlation.txt
"""
}
/*
* STEP 7 deepTools
*/
process deepTools {
tag "${bam[0].baseName}"
publishDir "${params.outdir}/deepTools", mode: 'copy'
input:
file bam from bam_dedup_deepTools.collect()
file bai from bai_dedup_deepTools.collect()
file bed from bed
output:
file '*.{txt,pdf,png,npz,bw}' into deepTools_results
file '*.txt' into deepTools_multiqc
script:
if (!params.singleEnd) {
"""
bamPEFragmentSize \\
--binSize 1000 \\
--bamfiles $bam \\
--o fragment_size_histogram.pdf \\
--plotFileFormat pdf \\
--plotTitle "Paired-end Fragment Size Distribution" \\
--outRawFragmentLengths bamPEFragmentSize_rawdata.txt
"""
}
if(bam instanceof Path){
log.warn("Only 1 BAM file - skipping multiBam deepTool steps")
"""
plotFingerprint \\
-b $bam \\
--plotFile fingerprints.pdf \\
--outRawCounts fingerprint.txt \\
--extendReads ${params.extendReadsLen} \\
--skipZeros \\
--ignoreDuplicates \\
--numberOfSamples 50000 \\
--binSize 500 \\
--plotFileFormat pdf \\
--plotTitle "Fingerprints"
bamCoverage \\
-b $bam \\
--extendReads ${params.extendReadsLen} \\
--normalizeUsing RPKM \\
-o ${bam}.bw
computeMatrix \\
scale-regions \\
--scoreFileName ${bam}.bw \\
--regionsFileName $bed \\
--beforeRegionStartLength 3000 \\
--afterRegionStartLength 3000 \\
--regionBodyLength 5000 \\
--outFileName computeMatrix.out.gz \\
--skipZeros \\
--smartLabels
plotProfile \\
--matrixFile computeMatrix.out.gz \\
--outFileName read_distribution_profile.pdf \\
--plotFileFormat pdf \\
--outFileNameData read_distribution_profile.txt \\
--plotTitle "Reads Distribution Profile"
"""
} else {
"""
plotFingerprint \\
-b $bam \\
--plotFile fingerprints.pdf \\
--outRawCounts fingerprint.txt \\
--extendReads ${params.extendReadsLen} \\
--skipZeros \\
--ignoreDuplicates \\
--numberOfSamples 50000 \\
--binSize 500 \\
--plotFileFormat pdf \\
--plotTitle "Fingerprints"
for bamfile in ${bam}
do
bamCoverage \\
-b \$bamfile \\
--extendReads ${params.extendReadsLen} \\
--normalizeUsing RPKM \\
-o \${bamfile}.bw
done
computeMatrix \\
scale-regions \\
--scoreFileName *.bw \\
--regionsFileName $bed \\
--beforeRegionStartLength 3000 \\
--afterRegionStartLength 3000 \\
--regionBodyLength 5000 \\
--outFileName computeMatrix.out.gz \\
--skipZeros \\
--smartLabels
plotProfile \\
--matrixFile computeMatrix.out.gz \\
--outFileName reads_distribution_profile.pdf \\
--plotFileFormat pdf \\
--outFileNameData read_distribution_profile.txt \\
--plotTitle "Reads Distribution Profile"
multiBamSummary \\
bins \\
--binSize 10000 \\
--bamfiles $bam \\
-out multiBamSummary.npz \\
--extendReads ${params.extendReadsLen} \\
--ignoreDuplicates \\
--centerReads \\
--smartLabels
plotCorrelation \\
-in multiBamSummary.npz \\
-o heatmap_SpearmanCorr.pdf \\
--plotFileFormat pdf \\
--outFileCorMatrix heatmap_SpearmanCorr.txt \\
--corMethod spearman \\
--skipZeros \\
--plotTitle "Spearman Correlation of Read Counts" \\
--whatToPlot heatmap \\
--colorMap RdYlBu \\
--plotNumbers
plotPCA \\
-in multiBamSummary.npz \\
-o pcaplot.pdf \\
--plotFileFormat pdf \\
--plotTitle "Principal Component Analysis Plot" \\
--outFileNameData pcaplot.txt \\
--plotWidth 8
"""
}
}
/*
* STEP 8.1 MACS
*/
process macs {
tag "${analysis_id}"
publishDir "${params.outdir}/macs", mode: 'copy'
input:
file bam from bam_dedup_macs.collect()
file bai from bai_dedup_macs.collect()
set chip_sample_id, ctrl_sample_id, analysis_id from macs_para
output:
file '*.{bed,r,narrowPeak}' into macs_results
file '*.xls' into macs_peaks
script:
if(!params.skipDupRemoval){
chip = "-t ${chip_sample_id}.dedup.sorted.bam"
ctrl = ctrl_sample_id == '' ? '' : "-c ${ctrl_sample_id}.dedup.sorted.bam"
}
else {
chip = "-t ${chip_sample_id}.sorted.bam"
ctrl = ctrl_sample_id == '' ? '' : "-c ${ctrl_sample_id}.sorted.bam"
}
broad = params.broad || params.ATACseq ? "--broad" : ''
"""
macs2 callpeak \\
$chip \\
$ctrl \\
$broad \\
-f BAM \\
-g $params.macsgsize \\
-n $analysis_id \\
-q 0.01
"""
}
/*
* STEP 8.2 Saturation analysis
*/
if (params.saturation) {
process saturation {
tag "${analysis_id}.${sampling}"
publishDir "${params.outdir}/macs/saturation", mode: 'copy'
input:
file bam from bam_dedup_saturation.collect()
file bai from bai_dedup_saturation.collect()
set chip_sample_id, ctrl_sample_id, analysis_id from saturation_para
each sampling from 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0
output:
file '*.xls' into saturation_results
script:
if(!params.skipDupRemoval){
chip_sample = "${chip_sample_id}.dedup.sorted.bam"
ctrl = ctrl_sample_id == '' ? '' : "-c ${ctrl_sample_id}.dedup.sorted.bam"
}
else {
chip_sample = "${chip_sample_id}.sorted.bam"
ctrl = ctrl_sample_id == '' ? '' : "-c ${ctrl_sample_id}.sorted.bam"
}
broad = params.broad || params.ATACseq ? "--broad" : ''
"""
samtools view -b -s ${sampling} ${chip_sample} > ${chip_sample}.${sampling}.bam
macs2 callpeak \\
-t ${chip_sample}.${sampling}.bam \\
$ctrl \\
$broad \\
-f BAM \\
-g $params.macsgsize \\
-n ${analysis_id}.${sampling} \\
-q 0.01
"""
}
process saturation_r {
tag "${saturation_results_collection[0].baseName}"
publishDir "${params.outdir}/macs/saturation", mode: 'copy'
input:
file macsconfig from macsconfig
file countstat from countstat_results
file saturation_results_collection from saturation_results.collect()
output:
file '*.{txt,pdf}' into saturation_summary
script:
"""
saturation_results_processing.r $params.rlocation $macsconfig $countstat $saturation_results_collection
"""
}
}
/*
* STEP 9 Post peak calling processing
*/
process chippeakanno {
tag "${macs_peaks_collection[0].baseName}"
publishDir "${params.outdir}/macs/chippeakanno", mode: 'copy'
input:
file macs_peaks_collection from macs_peaks.collect()
file gtf from gtf
output:
file '*.{txt,bed}' into chippeakanno_results
script:
filtering = params.blacklist_filtering ? "${params.blacklist}" : "No-filtering"
"""
post_peak_calling_processing.r $params.rlocation $filtering $gtf $macs_peaks_collection
"""
}
/*
* Parse software version numbers
*/
process get_software_versions {
output:
file 'software_versions_mqc.yaml' into software_versions_yaml
script:
"""
echo ${params.pipelineVersion} > v_ngi_chipseq.txt
echo $workflow.nextflow.version > v_nextflow.txt
fastqc --version > v_fastqc.txt
trim_galore --version > v_trim_galore.txt
echo \$(bwa 2>&1) > v_bwa.txt
samtools --version > v_samtools.txt
bedtools --version > v_bedtools.txt
echo "version" \$(java -Xmx2g -jar \$PICARD_HOME/picard.jar MarkDuplicates --version 2>&1) >v_picard.txt
echo \$(plotFingerprint --version 2>&1) > v_deeptools.txt
echo \$(macs2 --version 2>&1) > v_macs2.txt
multiqc --version > v_multiqc.txt
scrape_software_versions.py > software_versions_mqc.yaml
"""
}
/*
* STEP 10 MultiQC
*/
process multiqc {
tag "$prefix"
publishDir "${params.outdir}/MultiQC", mode: 'copy'
input:
file multiqc_config
file (fastqc:'fastqc/*') from fastqc_results.collect()
file ('trimgalore/*') from trimgalore_results.collect()
file ('samtools/*') from samtools_stats.collect()
file ('picard/*') from picard_reports.collect()
file ('deeptools/*') from deepTools_multiqc.collect()
file ('phantompeakqualtools/*') from spp_out_mqc.collect()
file ('phantompeakqualtools/*') from spp_csv_mqc.collect()
file ('software_versions/*') from software_versions_yaml.collect()
output:
file '*multiqc_report.html' into multiqc_report
file '*_data' into multiqc_data
file '.command.err' into multiqc_stderr
val prefix into multiqc_prefix