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main.nf
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#!/usr/bin/env nextflow
/*
========================================================================================
nf-core/mnaseseq
========================================================================================
nf-core/mnaseseq Analysis Pipeline.
#### Homepage / Documentation
https://github.com/nf-core/mnaseseq
----------------------------------------------------------------------------------------
*/
def helpMessage() {
log.info nfcoreHeader()
log.info"""
Usage:
The typical command for running the pipeline is as follows:
nextflow run nf-core/mnaseseq --input design.csv --genome GRCh37 -profile docker
Mandatory arguments:
--input [file] Comma-separated file containing information about the samples in the experiment (see docs/usage.md)
--fasta [file] Path to Fasta reference. Not mandatory when using reference in iGenomes config via --genome
--gtf [file] Path to GTF file. Not mandatory when using reference in iGenomes config via --genome
-profile [str] Configuration profile to use. Can use multiple (comma separated)
Available: conda, docker, singularity, awsbatch, test
Generic
--single_end [bool] Specifies that the input is single-end reads
--seq_center [str] Sequencing center information to be added to read group of BAM files
--fragment_size [int] Estimated fragment size used to extend single-end reads (Default: 150)
--fingerprint_bins [int] Number of genomic bins to use when calculating fingerprint plot (Default: 500000)
References If not specified in the configuration file or you wish to overwrite any of the references
--genome [str] Name of iGenomes reference
--bwa_index [file] Full path to directory containing BWA index including base name i.e. /path/to/index/genome.fa
--gene_bed [file] Path to BED file containing gene intervals
--tss_bed [file] Path to BED file containing transcription start sites
--blacklist [file] Path to blacklist regions (.BED format), used for filtering alignments
--save_reference [bool] If generated by the pipeline save the BWA index in the results directory
Trimming
--clip_r1 [int] Instructs Trim Galore to remove bp from the 5' end of read 1 (or single-end reads) (Default: 0)
--clip_r2 [int] Instructs Trim Galore to remove bp from the 5' end of read 2 (paired-end reads only) (Default: 0)
--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 (Default: 0)
--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 (Default: 0)
--trim_nextseq [int] Instructs Trim Galore to apply the --nextseq=X option, to trim based on quality after removing poly-G tails (Default: 0)
--skip_trimming [bool] Skip the adapter trimming step
--save_trimmed [bool] Save the trimmed FastQ files in the results directory
Alignments
--min_insert [int] Minimum insert size for filtering of mono-nucleosome paired-end reads (default: 100)
--max_insert [int] Maximum insert size for filtering of mono-nucleosome paired-end reads (default: 200)
--max_mismatch [int] Maximum number of mismatches in alignment. Derived from XM tag in BAM file (default: 4)
--keep_dups [bool] Duplicate reads are not filtered from alignments
--keep_multi_map [bool] Reads mapping to multiple locations are not filtered from alignments
--skip_merge_replicates [bool] Do not perform alignment merging and downstream analysis by merging replicates i.e. only do this by merging resequenced libraries
--save_align_intermeds [bool] Save the intermediate BAM files from the alignment step - not done by default
QC
--skip_fastqc [bool] Skip FastQC
--skip_picard_metrics [bool] Skip Picard CollectMultipleMetrics
--skip_preseq [bool] Skip Preseq
--skip_plot_profile [bool] Skip deepTools plotProfile
--skip_plot_fingerprint [bool] Skip deepTools plotFingerprint
--skip_danpos [bool] Skip DANPOS2
--skip_igv [bool] Skip IGV
--skip_multiqc [bool] Skip MultiQC
Other
--outdir [file] The output directory where the results will be saved
--email [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
--email_on_fail [email] Same as --email, except only send mail if the workflow is not successful
--max_multiqc_email_size [str] Theshold size for MultiQC report to be attached in notification email. If file generated by pipeline exceeds the threshold, it will not be attached (Default: 25MB)
-name [str] Name for the pipeline run. If not specified, Nextflow will automatically generate a random mnemonic
AWSBatch options:
--awsqueue [str] The AWSBatch JobQueue that needs to be set when running on AWSBatch
--awsregion [str] The AWS Region for your AWS Batch job to run on
--awscli [str] Path to the AWS CLI tool
""".stripIndent()
}
///////////////////////////////////////////////////////////////////////////////
///////////////////////////////////////////////////////////////////////////////
/* -- -- */
/* -- SET UP CONFIGURATION VARIABLES -- */
/* -- -- */
///////////////////////////////////////////////////////////////////////////////
///////////////////////////////////////////////////////////////////////////////
/*
* SET UP CONFIGURATION VARIABLES
*/
// Show help message
if (params.help) {
helpMessage()
exit 0
}
/*
* SET UP CONFIGURATION VARIABLES
*/
// Check if genome exists in the config file
if (params.genomes && params.genome && !params.genomes.containsKey(params.genome)) {
exit 1, "The provided genome '${params.genome}' is not available in the iGenomes file. Currently the available genomes are ${params.genomes.keySet().join(", ")}"
}
////////////////////////////////////////////////////
/* -- DEFAULT PARAMETER VALUES -- */
////////////////////////////////////////////////////
// Configurable variables
params.fasta = params.genome ? params.genomes[ params.genome ].fasta ?: false : false
params.bwa_index = params.genome ? params.genomes[ params.genome ].bwa ?: false : false
params.gtf = params.genome ? params.genomes[ params.genome ].gtf ?: false : false
params.gene_bed = params.genome ? params.genomes[ params.genome ].bed12 ?: false : false
params.blacklist = params.genome ? params.genomes[ params.genome ].blacklist ?: false : false
params.anno_readme = params.genome ? params.genomes[ params.genome ].readme ?: false : false
// Has the run name been specified by the user?
// this has the bonus effect of catching both -name and --name
custom_runName = params.name
if (!(workflow.runName ==~ /[a-z]+_[a-z]+/)) {
custom_runName = workflow.runName
}
////////////////////////////////////////////////////
/* -- CONFIG FILES -- */
////////////////////////////////////////////////////
// Pipeline config
ch_multiqc_config = file("$baseDir/assets/multiqc_config.yaml", checkIfExists: true)
ch_multiqc_custom_config = params.multiqc_config ? Channel.fromPath(params.multiqc_config, checkIfExists: true) : Channel.empty()
ch_output_docs = file("$baseDir/docs/output.md", checkIfExists: true)
// JSON files required by BAMTools for alignment filtering
if (params.single_end) {
ch_bamtools_filter_config = file(params.bamtools_filter_se_config, checkIfExists: true)
} else {
ch_bamtools_filter_config = file(params.bamtools_filter_pe_config, checkIfExists: true)
}
////////////////////////////////////////////////////
/* -- VALIDATE INPUTS -- */
////////////////////////////////////////////////////
// Validate inputs
if (params.input) { ch_input = file(params.input, checkIfExists: true) } else { exit 1, "Samples design file not specified!" }
if (params.gtf) { ch_gtf = file(params.gtf, checkIfExists: true) } else { exit 1, "GTF annotation file not specified!" }
if (params.gene_bed) { ch_gene_bed = file(params.gene_bed, checkIfExists: true) }
if (params.tss_bed) { ch_tss_bed = file(params.tss_bed, checkIfExists: true) }
if (params.blacklist) { ch_blacklist = Channel.fromPath(params.blacklist, checkIfExists: true) } else { ch_blacklist = Channel.empty() }
if (params.anno_readme && file(params.anno_readme).exists()) { ch_anno_readme = Channel.fromPath(params.anno_readme) } else { ch_anno_readme = Channel.empty() }
if (params.fasta) {
lastPath = params.fasta.lastIndexOf(File.separator)
bwa_base = params.fasta.substring(lastPath+1)
ch_fasta = file(params.fasta, checkIfExists: true)
} else {
exit 1, "Fasta file not specified!"
}
if (params.bwa_index) {
lastPath = params.bwa_index.lastIndexOf(File.separator)
bwa_dir = params.bwa_index.substring(0,lastPath+1)
bwa_base = params.bwa_index.substring(lastPath+1)
Channel
.fromPath(bwa_dir, checkIfExists: true)
.set { ch_bwa_index }
}
////////////////////////////////////////////////////
/* -- AWS -- */
////////////////////////////////////////////////////
if (workflow.profile.contains('awsbatch')) {
// AWSBatch sanity checking
if (!params.awsqueue || !params.awsregion) exit 1, "Specify correct --awsqueue and --awsregion parameters on AWSBatch!"
// Check outdir paths to be S3 buckets if running on AWSBatch
// related: https://github.com/nextflow-io/nextflow/issues/813
if (!params.outdir.startsWith('s3:')) exit 1, "Outdir not on S3 - specify S3 Bucket to run on AWSBatch!"
// Prevent trace files to be stored on S3 since S3 does not support rolling files.
if (params.tracedir.startsWith('s3:')) exit 1, "Specify a local tracedir or run without trace! S3 cannot be used for tracefiles."
}
///////////////////////////////////////////////////////////////////////////////
///////////////////////////////////////////////////////////////////////////////
/* -- -- */
/* -- HEADER LOG INFO -- */
/* -- -- */
///////////////////////////////////////////////////////////////////////////////
///////////////////////////////////////////////////////////////////////////////
// Header log info
log.info nfcoreHeader()
def summary = [:]
summary['Run Name'] = custom_runName ?: workflow.runName
summary['Data Type'] = params.single_end ? 'Single-End' : 'Paired-End'
summary['Design File'] = params.input
summary['Genome'] = params.genome ?: 'Not supplied'
summary['Fasta File'] = params.fasta
summary['GTF File'] = params.gtf
if (params.gene_bed) summary['Gene BED File'] = params.gene_bed
if (params.tss_bed) summary['TSS BED File'] = params.tss_bed
if (params.bwa_index) summary['BWA Index'] = params.bwa_index
if (params.blacklist) summary['Blacklist BED'] = params.blacklist
if (params.skip_trimming) {
summary['Trimming Step'] = 'Skipped'
} else {
summary['Trim R1'] = "$params.clip_r1 bp"
summary['Trim R2'] = "$params.clip_r2 bp"
summary["Trim 3' R1"] = "$params.three_prime_clip_r1 bp"
summary["Trim 3' R2"] = "$params.three_prime_clip_r2 bp"
summary["NextSeq Trim"] = "$params.trim_nextseq bp"
}
if (params.seq_center) summary['Sequencing Center'] = params.seq_center
if (params.single_end) summary['Fragment Size'] = "$params.fragment_size bp"
summary['Fingerprint Bins'] = params.fingerprint_bins
summary['Max Mismatch'] = params.max_mismatch
summary['Min Insert Size'] = "$params.min_insert bp"
summary['Max Insert Size'] = "$params.max_insert bp"
if (params.keep_dups) summary['Keep Duplicates'] = 'Yes'
if (params.keep_multi_map) summary['Keep Multi-mapped'] = 'Yes'
summary['Save Genome Index'] = params.save_reference ? 'Yes' : 'No'
if (params.save_trimmed) summary['Save Trimmed'] = 'Yes'
if (params.save_align_intermeds) summary['Save Intermeds'] = 'Yes'
if (params.skip_merge_replicates) summary['Skip Merge Replicates'] = 'Yes'
if (params.skip_fastqc) summary['Skip FastQC'] = 'Yes'
if (params.skip_picard_metrics) summary['Skip Picard Metrics'] = 'Yes'
if (params.skip_preseq) summary['Skip Preseq'] = 'Yes'
if (params.skip_plot_profile) summary['Skip plotProfile'] = 'Yes'
if (params.skip_plot_fingerprint) summary['Skip plotFingerprint'] = 'Yes'
if (params.skip_danpos) summary['Skip DANPOS2'] = 'Yes'
if (params.skip_igv) summary['Skip IGV'] = 'Yes'
if (params.skip_multiqc) summary['Skip MultiQC'] = 'Yes'
summary['Max Resources'] = "$params.max_memory memory, $params.max_cpus cpus, $params.max_time time per job"
if (workflow.containerEngine) summary['Container'] = "$workflow.containerEngine - $workflow.container"
summary['Output Dir'] = params.outdir
summary['Launch Dir'] = workflow.launchDir
summary['Working Dir'] = workflow.workDir
summary['Script Dir'] = workflow.projectDir
summary['User'] = workflow.userName
if (workflow.profile.contains('awsbatch')) {
summary['AWS Region'] = params.awsregion
summary['AWS Queue'] = params.awsqueue
summary['AWS CLI'] = params.awscli
}
summary['Config Profile'] = workflow.profile
if (params.config_profile_description) summary['Config Description'] = params.config_profile_description
if (params.config_profile_contact) summary['Config Contact'] = params.config_profile_contact
if (params.config_profile_url) summary['Config URL'] = params.config_profile_url
if (params.email || params.email_on_fail) {
summary['E-mail Address'] = params.email
summary['E-mail on failure'] = params.email_on_fail
summary['MultiQC Max Size'] = params.max_multiqc_email_size
}
log.info summary.collect { k,v -> "${k.padRight(21)}: $v" }.join("\n")
log.info "-\033[2m--------------------------------------------------\033[0m-"
// Check the hostnames against configured profiles
checkHostname()
///////////////////////////////////////////////////////////////////////////////
///////////////////////////////////////////////////////////////////////////////
/* -- -- */
/* -- PARSE DESIGN FILE -- */
/* -- -- */
///////////////////////////////////////////////////////////////////////////////
///////////////////////////////////////////////////////////////////////////////
/*
* PREPROCESSING - REFORMAT DESIGN FILE AND CHECK VALIDITY
*/
process CheckDesign {
tag "$design"
publishDir "${params.outdir}/pipeline_info", mode: 'copy'
input:
file design from ch_input
output:
file "*.csv" into ch_design_reads_csv
script: // This script is bundled with the pipeline, in nf-core/mnaseseq/bin/
"""
check_design.py $design design_reads.csv
"""
}
/*
* Create channels for input fastq files
*/
if (params.single_end) {
ch_design_reads_csv
.splitCsv(header:true, sep:',')
.map { row -> [ row.sample_id, [ file(row.fastq_1, checkIfExists: true) ] ] }
.into { ch_raw_reads_fastqc;
ch_raw_reads_trimgalore;
ch_design_replicates_exist }
} else {
ch_design_reads_csv
.splitCsv(header:true, sep:',')
.map { row -> [ row.sample_id, [ file(row.fastq_1, checkIfExists: true), file(row.fastq_2, checkIfExists: true) ] ] }
.into { ch_raw_reads_fastqc;
ch_raw_reads_trimgalore;
ch_design_replicates_exist }
}
// Boolean value for replicates existing in design
replicatesExist = ch_design_replicates_exist
.map { it -> it[0].split('_')[-2].replaceAll('R','').toInteger() }
.flatten()
.max()
.val > 1
///////////////////////////////////////////////////////////////////////////////
///////////////////////////////////////////////////////////////////////////////
/* -- -- */
/* -- PREPARE ANNOTATION FILES -- */
/* -- -- */
///////////////////////////////////////////////////////////////////////////////
///////////////////////////////////////////////////////////////////////////////
/*
* PREPROCESSING - Build BWA index
*/
if (!params.bwa_index) {
process BWAIndex {
tag "$fasta"
label 'process_high'
publishDir path: { params.save_reference ? "${params.outdir}/reference_genome" : params.outdir },
saveAs: { params.save_reference ? it : null }, mode: 'copy'
input:
file fasta from ch_fasta
output:
file "BWAIndex" into ch_bwa_index
script:
"""
bwa index -a bwtsw $fasta
mkdir BWAIndex && mv ${fasta}* BWAIndex
"""
}
}
/*
* PREPROCESSING - Generate gene BED file
*/
if (!params.gene_bed) {
process MakeGeneBED {
tag "$gtf"
label 'process_low'
publishDir "${params.outdir}/reference_genome", mode: 'copy'
input:
file gtf from ch_gtf
output:
file "*.bed" into ch_gene_bed
script: // This script is bundled with the pipeline, in nf-core/mnaseseq/bin/
"""
gtf2bed $gtf > ${gtf.baseName}.bed
"""
}
}
/*
* PREPROCESSING - Generate TSS BED file
*/
if (!params.tss_bed) {
process MakeTSSBED {
tag "$bed"
publishDir "${params.outdir}/reference_genome", mode: 'copy'
input:
file bed from ch_gene_bed
output:
file "*.bed" into ch_tss_bed
script:
"""
cat $bed | awk -v FS='\t' -v OFS='\t' '{ if(\$6=="+") \$3=\$2+1; else \$2=\$3-1; print \$1, \$2, \$3, \$4, \$5, \$6;}' > ${bed.baseName}.tss.bed
"""
}
}
/*
* PREPROCESSING - Prepare genome intervals for filtering
*/
process MakeGenomeFilter {
tag "$fasta"
publishDir "${params.outdir}/reference_genome", mode: 'copy'
input:
file fasta from ch_fasta
file blacklist from ch_blacklist.ifEmpty([])
file readme from ch_anno_readme.ifEmpty([])
output:
file "$fasta" // FASTA FILE FOR IGV
//file "$readme" // AWS IGENOMES FILE CONTAINING ANNOTATION VERSION
file "*.fai" // FAI INDEX FOR REFERENCE GENOME
file "*.bed" into ch_genome_filter_regions // BED FILE WITHOUT BLACKLIST REGIONS & MITOCHONDRIAL CONTIG FOR FILTERING
file "*.sizes" into ch_genome_sizes_mlib_bigwig, // CHROMOSOME SIZES FILE FOR BEDTOOLS
ch_genome_sizes_mrep_bigwig,
ch_genome_sizes_mlib_danpos,
ch_genome_sizes_mrep_danpos
script:
blacklist_filter = params.blacklist ? "sortBed -i $blacklist -g ${fasta}.sizes | complementBed -i stdin -g ${fasta}.sizes" : "awk '{print \$1, '0' , \$2}' OFS='\t' ${fasta}.sizes"
"""
samtools faidx $fasta
cut -f 1,2 ${fasta}.fai > ${fasta}.sizes
$blacklist_filter > ${fasta}.include_regions.bed
"""
}
///////////////////////////////////////////////////////////////////////////////
///////////////////////////////////////////////////////////////////////////////
/* -- -- */
/* -- FASTQ QC -- */
/* -- -- */
///////////////////////////////////////////////////////////////////////////////
///////////////////////////////////////////////////////////////////////////////
/*
* STEP 1 - FastQC
*/
process FastQC {
tag "$name"
label 'process_medium'
publishDir "${params.outdir}/fastqc", mode: 'copy',
saveAs: { filename ->
filename.endsWith(".zip") ? "zips/$filename" : "$filename"
}
when:
!params.skip_fastqc
input:
set val(name), file(reads) from ch_raw_reads_fastqc
output:
file "*.{zip,html}" into ch_fastqc_reports_mqc
script:
// Added soft-links to original fastqs for consistent naming in MultiQC
if (params.single_end) {
"""
[ ! -f ${name}.fastq.gz ] && ln -s $reads ${name}.fastq.gz
fastqc -q -t $task.cpus ${name}.fastq.gz
"""
} else {
"""
[ ! -f ${name}_1.fastq.gz ] && ln -s ${reads[0]} ${name}_1.fastq.gz
[ ! -f ${name}_2.fastq.gz ] && ln -s ${reads[1]} ${name}_2.fastq.gz
fastqc -q -t $task.cpus ${name}_1.fastq.gz
fastqc -q -t $task.cpus ${name}_2.fastq.gz
"""
}
}
///////////////////////////////////////////////////////////////////////////////
///////////////////////////////////////////////////////////////////////////////
/* -- -- */
/* -- ADAPTER TRIMMING -- */
/* -- -- */
///////////////////////////////////////////////////////////////////////////////
///////////////////////////////////////////////////////////////////////////////
/*
* STEP 2 - Trim Galore!
*/
if (params.skip_trimming) {
ch_trimmed_reads = ch_raw_reads_trimgalore
ch_trimgalore_results_mqc = Channel.empty()
ch_trimgalore_fastqc_reports_mqc = Channel.empty()
} else {
process TrimGalore {
tag "$name"
label 'process_high'
publishDir "${params.outdir}/trim_galore", mode: 'copy',
saveAs: { filename ->
if (filename.endsWith(".html")) "fastqc/$filename"
else if (filename.endsWith(".zip")) "fastqc/zips/$filename"
else if (filename.endsWith("trimming_report.txt")) "logs/$filename"
else params.save_trimmed ? filename : null
}
input:
set val(name), file(reads) from ch_raw_reads_trimgalore
output:
set val(name), file("*.fq.gz") into ch_trimmed_reads
file "*.txt" into ch_trimgalore_results_mqc
file "*.{zip,html}" into ch_trimgalore_fastqc_reports_mqc
script:
// Calculate number of --cores for TrimGalore based on value of task.cpus
// See: https://github.com/FelixKrueger/TrimGalore/blob/master/Changelog.md#version-060-release-on-1-mar-2019
// See: https://github.com/nf-core/atacseq/pull/65
def cores = 1
if (task.cpus) {
cores = (task.cpus as int) - 4
if (params.single_end) cores = (task.cpus as int) - 3
if (cores < 1) cores = 1
if (cores > 4) cores = 4
}
// Added soft-links to original fastqs for consistent naming in MultiQC
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}" : ''
nextseq = params.trim_nextseq > 0 ? "--nextseq ${params.trim_nextseq}" : ''
if (params.single_end) {
"""
[ ! -f ${name}.fastq.gz ] && ln -s $reads ${name}.fastq.gz
trim_galore --cores $cores --fastqc --gzip $c_r1 $tpc_r1 $nextseq ${name}.fastq.gz
"""
} else {
"""
[ ! -f ${name}_1.fastq.gz ] && ln -s ${reads[0]} ${name}_1.fastq.gz
[ ! -f ${name}_2.fastq.gz ] && ln -s ${reads[1]} ${name}_2.fastq.gz
trim_galore --cores $cores --paired --fastqc --gzip $c_r1 $c_r2 $tpc_r1 $tpc_r2 $nextseq ${name}_1.fastq.gz ${name}_2.fastq.gz
"""
}
}
}
///////////////////////////////////////////////////////////////////////////////
///////////////////////////////////////////////////////////////////////////////
/* -- -- */
/* -- ALIGN -- */
/* -- -- */
///////////////////////////////////////////////////////////////////////////////
///////////////////////////////////////////////////////////////////////////////
/*
* STEP 3.1 - Align read 1 with bwa
*/
process BWAMem {
tag "$name"
label 'process_high'
input:
set val(name), file(reads) from ch_trimmed_reads
file index from ch_bwa_index.collect()
output:
set val(name), file("*.bam") into ch_bwa_bam
script:
prefix = "${name}.Lb"
rg = "\'@RG\\tID:${name}\\tSM:${name.split('_')[0..-2].join('_')}\\tPL:ILLUMINA\\tLB:${name}\\tPU:1\'"
if (params.seq_center) {
rg = "\'@RG\\tID:${name}\\tSM:${name.split('_')[0..-2].join('_')}\\tPL:ILLUMINA\\tLB:${name}\\tPU:1\\tCN:${params.seq_center}\'"
}
"""
bwa mem \\
-t $task.cpus \\
-M \\
-R $rg \\
${index}/${bwa_base} \\
$reads \\
| samtools view -@ $task.cpus -b -h -F 0x0100 -O BAM -o ${prefix}.bam -
"""
}
/*
* STEP 3.2 - Convert .bam to coordinate sorted .bam
*/
process SortBAM {
tag "$name"
label 'process_medium'
if (params.save_align_intermeds) {
publishDir path: "${params.outdir}/bwa/library", mode: 'copy',
saveAs: { filename ->
if (filename.endsWith(".flagstat")) "samtools_stats/$filename"
else if (filename.endsWith(".idxstats")) "samtools_stats/$filename"
else if (filename.endsWith(".stats")) "samtools_stats/$filename"
else filename
}
}
input:
set val(name), file(bam) from ch_bwa_bam
output:
set val(name), file("*.sorted.{bam,bam.bai}") into ch_sort_bam_merge
file "*.{flagstat,idxstats,stats}" into ch_sort_bam_flagstat_mqc
script:
prefix = "${name}.Lb"
"""
samtools sort -@ $task.cpus -o ${prefix}.sorted.bam -T $name $bam
samtools index ${prefix}.sorted.bam
samtools flagstat ${prefix}.sorted.bam > ${prefix}.sorted.bam.flagstat
samtools idxstats ${prefix}.sorted.bam > ${prefix}.sorted.bam.idxstats
samtools stats ${prefix}.sorted.bam > ${prefix}.sorted.bam.stats
"""
}
///////////////////////////////////////////////////////////////////////////////
///////////////////////////////////////////////////////////////////////////////
/* -- -- */
/* -- MERGE LIBRARY BAM -- */
/* -- -- */
///////////////////////////////////////////////////////////////////////////////
///////////////////////////////////////////////////////////////////////////////
/*
* STEP 4.1 Merge BAM files for all libraries from same replicate
*/
ch_sort_bam_merge
.map { it -> [ it[0].split('_')[0..-2].join('_'), it[1] ] }
.groupTuple(by: [0])
.map { it -> [ it[0], it[1].flatten() ] }
.set { ch_sort_bam_merge }
process MergedLibBAM {
tag "$name"
label 'process_medium'
publishDir "${params.outdir}/bwa/mergedLibrary", mode: 'copy',
saveAs: { filename ->
if (filename.endsWith(".flagstat")) "samtools_stats/$filename"
else if (filename.endsWith(".idxstats")) "samtools_stats/$filename"
else if (filename.endsWith(".stats")) "samtools_stats/$filename"
else if (filename.endsWith(".metrics.txt")) "picard_metrics/$filename"
else params.save_align_intermeds ? filename : null
}
input:
set val(name), file(bams) from ch_sort_bam_merge
output:
set val(name), file("*${prefix}.sorted.{bam,bam.bai}") into ch_mlib_bam_filter,
ch_mlib_bam_preseq,
ch_mlib_bam_metrics
file "*.{flagstat,idxstats,stats}" into ch_mlib_bam_stats_mqc
file "*.txt" into ch_mlib_bam_metrics_mqc
script:
prefix = "${name}.mLb.mkD"
bam_files = bams.findAll { it.toString().endsWith('.bam') }.sort()
def avail_mem = 3
if (!task.memory) {
log.info "[Picard MarkDuplicates] Available memory not known - defaulting to 3GB. Specify process memory requirements to change this."
} else {
avail_mem = task.memory.toGiga()
}
if (bam_files.size() > 1) {
"""
picard -Xmx${avail_mem}g MergeSamFiles \\
${'INPUT='+bam_files.join(' INPUT=')} \\
OUTPUT=${name}.sorted.bam \\
SORT_ORDER=coordinate \\
VALIDATION_STRINGENCY=LENIENT \\
TMP_DIR=tmp
samtools index ${name}.sorted.bam
picard -Xmx${avail_mem}g MarkDuplicates \\
INPUT=${name}.sorted.bam \\
OUTPUT=${prefix}.sorted.bam \\
ASSUME_SORTED=true \\
REMOVE_DUPLICATES=false \\
METRICS_FILE=${prefix}.MarkDuplicates.metrics.txt \\
VALIDATION_STRINGENCY=LENIENT \\
TMP_DIR=tmp
samtools index ${prefix}.sorted.bam
samtools idxstats ${prefix}.sorted.bam > ${prefix}.sorted.bam.idxstats
samtools flagstat ${prefix}.sorted.bam > ${prefix}.sorted.bam.flagstat
samtools stats ${prefix}.sorted.bam > ${prefix}.sorted.bam.stats
"""
} else {
"""
picard -Xmx${avail_mem}g MarkDuplicates \\
INPUT=${bam_files[0]} \\
OUTPUT=${prefix}.sorted.bam \\
ASSUME_SORTED=true \\
REMOVE_DUPLICATES=false \\
METRICS_FILE=${prefix}.MarkDuplicates.metrics.txt \\
VALIDATION_STRINGENCY=LENIENT \\
TMP_DIR=tmp
samtools index ${prefix}.sorted.bam
samtools idxstats ${prefix}.sorted.bam > ${prefix}.sorted.bam.idxstats
samtools flagstat ${prefix}.sorted.bam > ${prefix}.sorted.bam.flagstat
samtools stats ${prefix}.sorted.bam > ${prefix}.sorted.bam.stats
"""
}
}
/*
* STEP 4.2 Filter BAM file at merged library-level
*/
process MergedLibBAMFilter {
tag "$name"
label 'process_medium'
publishDir path: "${params.outdir}/bwa/mergedLibrary", mode: 'copy',
saveAs: { filename ->
if (params.single_end || params.save_align_intermeds) {
if (filename.endsWith(".flagstat")) "samtools_stats/$filename"
else if (filename.endsWith(".idxstats")) "samtools_stats/$filename"
else if (filename.endsWith(".stats")) "samtools_stats/$filename"
else if (filename.endsWith(".sorted.bam")) filename
else if (filename.endsWith(".sorted.bam.bai")) filename
else null
}
}
input:
set val(name), file(bam) from ch_mlib_bam_filter
file bed from ch_genome_filter_regions.collect()
file bamtools_filter_config from ch_bamtools_filter_config
output:
set val(name), file("*.{bam,bam.bai}") into ch_mlib_filter_bam
set val(name), file("*.flagstat") into ch_mlib_filter_bam_flagstat
file "*.{idxstats,stats}" into ch_mlib_filter_bam_stats_mqc
script:
prefix = params.single_end ? "${name}.mLb.clN" : "${name}.mLb.flT"
filter_params = params.single_end ? "-F 0x004" : "-F 0x004 -F 0x0008 -f 0x001"
dup_params = params.keep_dups ? "" : "-F 0x0400"
multimap_params = params.keep_multi_map ? "" : "-q 1"
blacklist_params = params.blacklist ? "-L $bed" : ""
name_sort_bam = params.single_end ? "" : "samtools sort -n -@ $task.cpus -o ${prefix}.bam -T $prefix ${prefix}.sorted.bam"
"""
sed 's/MIN_INSERT_SIZE/${params.min_insert}/g' <$bamtools_filter_config >bamtools_filter.json
sed -i -e 's/MAX_INSERT_SIZE/${params.max_insert}/g' bamtools_filter.json
sed -i -e 's/MAX_MISMATCH/${params.max_mismatch}/g' bamtools_filter.json
samtools view \\
$filter_params \\
$dup_params \\
$multimap_params \\
$blacklist_params \\
-b ${bam[0]} \\
| bamtools filter \\
-out ${prefix}.sorted.bam \\
-script bamtools_filter.json
samtools index ${prefix}.sorted.bam
samtools flagstat ${prefix}.sorted.bam > ${prefix}.sorted.bam.flagstat
samtools idxstats ${prefix}.sorted.bam > ${prefix}.sorted.bam.idxstats
samtools stats ${prefix}.sorted.bam > ${prefix}.sorted.bam.stats
$name_sort_bam
"""
}
/*
* STEP 4.3 Remove orphan reads from paired-end BAM file
*/
if (params.single_end) {
ch_mlib_filter_bam
.into { ch_mlib_rm_orphan_bam_metrics;
ch_mlib_rm_orphan_bam_bigwig;
ch_mlib_rm_orphan_bam_plotfingerprint;
ch_mlib_rm_orphan_bam_mrep;
ch_mlib_name_bam_danpos }
ch_mlib_filter_bam_flagstat
.into { ch_mlib_rm_orphan_flagstat_bigwig;
ch_mlib_rm_orphan_flagstat_mqc }
ch_mlib_filter_bam_stats_mqc
.set { ch_mlib_rm_orphan_stats_mqc }
} else {
process MergedLibBAMRemoveOrphan {
tag "$name"
label 'process_medium'
publishDir path: "${params.outdir}/bwa/mergedLibrary", mode: 'copy',
saveAs: { filename ->
if (filename.endsWith(".flagstat")) "samtools_stats/$filename"
else if (filename.endsWith(".idxstats")) "samtools_stats/$filename"
else if (filename.endsWith(".stats")) "samtools_stats/$filename"
else if (filename.endsWith(".sorted.bam")) filename
else if (filename.endsWith(".sorted.bam.bai")) filename
else null
}
input:
set val(name), file(bam) from ch_mlib_filter_bam
output:
set val(name), file("*.sorted.{bam,bam.bai}") into ch_mlib_rm_orphan_bam_metrics,
ch_mlib_rm_orphan_bam_bigwig,
ch_mlib_rm_orphan_bam_plotfingerprint,
ch_mlib_rm_orphan_bam_mrep
set val(name), file("${prefix}.bam") into ch_mlib_name_bam_danpos
set val(name), file("*.flagstat") into ch_mlib_rm_orphan_flagstat_bigwig,
ch_mlib_rm_orphan_flagstat_mqc
file "*.{idxstats,stats}" into ch_mlib_rm_orphan_stats_mqc
script: // This script is bundled with the pipeline, in nf-core/mnaseseq/bin/
prefix = "${name}.mLb.clN"
"""
bampe_rm_orphan.py ${bam[0]} ${prefix}.bam --only_fr_pairs
samtools sort -@ $task.cpus -o ${prefix}.sorted.bam -T $prefix ${prefix}.bam
samtools index ${prefix}.sorted.bam
samtools flagstat ${prefix}.sorted.bam > ${prefix}.sorted.bam.flagstat
samtools idxstats ${prefix}.sorted.bam > ${prefix}.sorted.bam.idxstats
samtools stats ${prefix}.sorted.bam > ${prefix}.sorted.bam.stats
"""
}
}
///////////////////////////////////////////////////////////////////////////////
///////////////////////////////////////////////////////////////////////////////
/* -- -- */
/* -- MERGE LIBRARY BAM POST-ANALYSIS -- */
/* -- -- */
///////////////////////////////////////////////////////////////////////////////
///////////////////////////////////////////////////////////////////////////////
/*
* STEP 5.1 Preseq analysis after merging libraries and before filtering
*/
process MergedLibPreseq {
tag "$name"
label 'process_low'
publishDir "${params.outdir}/bwa/mergedLibrary/preseq", mode: 'copy'
when:
!params.skip_preseq
input:
set val(name), file(bam) from ch_mlib_bam_preseq
output:
file "*.ccurve.txt" into ch_mlib_preseq_mqc
script:
prefix = "${name}.mLb.mkD"
"""
preseq lc_extrap -v -output ${prefix}.ccurve.txt -bam ${bam[0]}
"""
}
/*
* STEP 5.2 Picard CollectMultipleMetrics after merging libraries and before filtering
*/
process MergedLibMetricsPreFilter {
tag "$name"
label 'process_medium'
publishDir path: "${params.outdir}/bwa/mergedLibrary", mode: 'copy',
saveAs: { filename ->
if (filename.endsWith("_metrics")) "picard_metrics/$filename"
else if (filename.endsWith(".pdf")) "picard_metrics/pdf/$filename"
else null
}
when:
!params.skip_picard_metrics
input:
set val(name), file(bam) from ch_mlib_bam_metrics
file fasta from ch_fasta
output:
file "*.pdf"
file "*_metrics" into ch_mlib_collectmetrics_prefilter_mqc
script:
prefix="${name}.mLb.mkD"
def avail_mem = 3
if (!task.memory) {
log.info "[Picard MarkDuplicates] Available memory not known - defaulting to 3GB. Specify process memory requirements to change this."
} else {
avail_mem = task.memory.toGiga()
}
"""
picard -Xmx${avail_mem}g CollectMultipleMetrics \\
INPUT=${bam[0]} \\
OUTPUT=${prefix}.CollectMultipleMetrics \\
REFERENCE_SEQUENCE=$fasta \\
VALIDATION_STRINGENCY=LENIENT \\
TMP_DIR=tmp
"""
}
/*
* STEP 5.3 Picard CollectMultipleMetrics after merging libraries and filtering
*/
process MergedLibMetricsPostFilter {
tag "$name"
label 'process_medium'
publishDir path: "${params.outdir}/bwa/mergedLibrary", mode: 'copy',
saveAs: { filename ->
if (filename.endsWith("_metrics")) "picard_metrics/$filename"
else if (filename.endsWith(".pdf")) "picard_metrics/pdf/$filename"
else null
}
when:
!params.skip_picard_metrics
input:
set val(name), file(bam) from ch_mlib_rm_orphan_bam_metrics
file fasta from ch_fasta
output:
file "*.pdf"
file "*_metrics" into ch_mlib_collectmetrics_postfilter_mqc
script:
prefix = "${name}.mLb.clN"
def avail_mem = 3
if (!task.memory) {
log.info "[Picard MarkDuplicates] Available memory not known - defaulting to 3GB. Specify process memory requirements to change this."
} else {
avail_mem = task.memory.toGiga()
}
"""
picard -Xmx${avail_mem}g CollectMultipleMetrics \\
INPUT=${bam[0]} \\
OUTPUT=${prefix}.CollectMultipleMetrics \\
REFERENCE_SEQUENCE=$fasta \\
VALIDATION_STRINGENCY=LENIENT \\
TMP_DIR=tmp
"""
}
/*
* STEP 5.4 Read depth normalised bigWig
*/
process MergedLibBigWig {
tag "$name"
label 'process_medium'
publishDir "${params.outdir}/bwa/mergedLibrary/bigwig", mode: 'copy',
saveAs: { filename ->
if (filename.endsWith("scale_factor.txt")) "scale/$filename"
else if (filename.endsWith(".bigWig")) "$filename"
else null
}
input:
set val(name), file(bam), file(flagstat) from ch_mlib_rm_orphan_bam_bigwig.join(ch_mlib_rm_orphan_flagstat_bigwig, by: [0])
file sizes from ch_genome_sizes_mlib_bigwig.collect()
output:
file "*.bigWig"
file "*scale_factor.txt"
file "*igv.txt" into ch_mlib_bigwig_igv
script:
prefix = "${name}.mLb.clN"
pe_fragment = params.single_end ? "" : "-pc"
extend = (params.single_end && params.fragment_size > 0) ? "-fs ${params.fragment_size}" : ''
"""
SCALE_FACTOR=\$(grep 'mapped (' $flagstat | awk '{print 1000000/\$1}')
echo \$SCALE_FACTOR > ${prefix}.scale_factor.txt
genomeCoverageBed -ibam ${bam[0]} -bg -scale \$SCALE_FACTOR $pe_fragment $extend | sort -T '.' -k1,1 -k2,2n > ${prefix}.bedGraph
bedGraphToBigWig ${prefix}.bedGraph $sizes ${prefix}.bigWig
find * -type f -name "*.bigWig" -exec echo -e "bwa/mergedLibrary/bigwig/"{}"\\t0,0,178" \\; > ${prefix}.bigWig.igv.txt
"""
}
/*
* STEP 5.5 deepTools plotFingerprint
*/
process MergedLibPlotFingerprint {
tag "$name"
label 'process_high'
publishDir "${params.outdir}/bwa/mergedLibrary/deepTools/plotFingerprint", mode: 'copy'
when:
!params.skip_plot_fingerprint
input:
set val(name), file(bam) from ch_mlib_rm_orphan_bam_plotfingerprint
output:
file '*.{txt,pdf}'
file '*.raw.txt' into ch_mlib_plotfingerprint_mqc
script:
prefix = "${name}.mLb.clN"
extend = (params.single_end && params.fragment_size > 0) ? "--extendReads ${params.fragment_size}" : ''
"""
plotFingerprint \\
--bamfiles ${bam[0]} \\
--plotFile ${prefix}.plotFingerprint.pdf \\
$extend \\
--labels $prefix \\
--outRawCounts ${prefix}.plotFingerprint.raw.txt \\
--outQualityMetrics ${prefix}.plotFingerprint.qcmetrics.txt \\