#!/usr/bin/env nextflow // Path to an input file, or a pattern for multiple inputs // Note - $baseDir is the location of this workflow file main.nf // Define Input variables params.reads = "$baseDir/../test_data/*.fastq.gz" params.pairedEnd = false params.designFile = "$baseDir/../test_data/design_ENCSR238SGC_SE.txt" params.genome = 'GRCm38' params.genomes = [] params.bwaIndex = params.genome ? params.genomes[ params.genome ].bwa ?: false : false params.genomeSize = params.genome ? params.genomes[ params.genome ].genomesize ?: false : false params.chromSizes = params.genome ? params.genomes[ params.genome ].chromsizes ?: false : false params.cutoffRatio = 1.2 // Check inputs if( params.bwaIndex ){ bwaIndex = Channel .fromPath(params.bwaIndex) .ifEmpty { exit 1, "BWA index not found: ${params.bwaIndex}" } } else { exit 1, "No reference genome specified." } // Define List of Files readsList = Channel .fromPath( params.reads ) .flatten() .map { file -> [ file.getFileName().toString(), file.toString() ].join("\t")} .collectFile( name: 'fileList.tsv', newLine: true ) // Define regular variables pairedEnd = params.pairedEnd designFile = params.designFile genomeSize = params.genomeSize chromSizes = params.chromSizes cutoffRatio = params.cutoffRatio process checkDesignFile { publishDir "$baseDir/output/design", mode: 'copy' input: designFile file readsList output: file("design.tsv") into designFilePaths script: if (pairedEnd) { """ python3 $baseDir/scripts/check_design.py -d $designFile -f $readsList -p """ } else { """ python $baseDir/scripts/check_design.py -d $designFile -f $readsList """ } } // Define channel for raw reads if (pairedEnd) { rawReads = designFilePaths .splitCsv(sep: '\t', header: true) .map { row -> [ row.sample_id, [row.fastq_read1, row.fastq_read2], row.experiment_id, row.biosample, row.factor, row.treatment, row.replicate, row.control_id ] } } else { rawReads = designFilePaths .splitCsv(sep: '\t', header: true) .map { row -> [ row.sample_id, [row.fastq_read1], row.experiment_id, row.biosample, row.factor, row.treatment, row.replicate, row.control_id ] } } // Trim raw reads using trimgalore process trimReads { tag "$sampleId-$replicate" publishDir "$baseDir/output/${task.process}", mode: 'copy' input: set sampleId, reads, experimentId, biosample, factor, treatment, replicate, controlId from rawReads output: set sampleId, file('*.fq.gz'), experimentId, biosample, factor, treatment, replicate, controlId into trimmedReads file('*trimming_report.txt') into trimgalore_results script: if (pairedEnd) { """ python3 $baseDir/scripts/trim_reads.py -f ${reads[0]} ${reads[1]} -p """ } else { """ python3 $baseDir/scripts/trim_reads.py -f ${reads[0]} """ } } // Align trimmed reads using bwa process alignReads { tag "$sampleId-$replicate" publishDir "$baseDir/output/${task.process}", mode: 'copy' input: set sampleId, reads, experimentId, biosample, factor, treatment, replicate, controlId from trimmedReads file index from bwaIndex.first() output: set sampleId, file('*.bam'), experimentId, biosample, factor, treatment, replicate, controlId into mappedReads file '*.srt.bam.flagstat.qc' into mappedReadsStats script: if (pairedEnd) { """ python3 $baseDir/scripts/map_reads.py -f ${reads[0]} ${reads[1]} -r ${index}/genome.fa -p """ } else { """ python3 $baseDir/scripts/map_reads.py -f $reads -r ${index}/genome.fa """ } } // Dedup reads using sambamba process filterReads { tag "$sampleId-$replicate" publishDir "$baseDir/output/${task.process}", mode: 'copy' input: set sampleId, mapped, experimentId, biosample, factor, treatment, replicate, controlId from mappedReads output: set sampleId, file('*.bam'), file('*.bai'), experimentId, biosample, factor, treatment, replicate, controlId into dedupReads set sampleId, file('*.bam'), experimentId, biosample, factor, treatment, replicate, controlId into convertReads file '*flagstat.qc' into dedupReadsStats file '*pbc.qc' into dedupReadsComplexity file '*dup.qc' into dupReads script: if (pairedEnd) { """ python3 $baseDir/scripts/map_qc.py -b $mapped -p """ } else { """ python3 $baseDir/scripts/map_qc.py -b $mapped """ } } // Define channel collecting dedup reads into new design file dedupReads .map{ sampleId, bam, bai, experimentId, biosample, factor, treatment, replicate, controlId -> "$sampleId\t$bam\t$bai\t$experimentId\t$biosample\t$factor\t$treatment\t$replicate\t$controlId\n"} .collectFile(name:'design_dedup.tsv', seed:"sample_id\tbam_reads\tbam_index\texperiment_id\tbiosample\tfactor\ttreatment\treplicate\tcontrol_id\n", storeDir:"$baseDir/output/design") .into { dedupDesign; preDiffDesign } // Quality Metrics using deeptools process experimentQC { publishDir "$baseDir/output/${task.process}", mode: 'copy' input: file dedupDesign output: file '*.{png,npz}' into deepToolsStats script: """ python3 $baseDir/scripts/experiment_qc.py -d $dedupDesign """ } // Convert reads to bam process convertReads { tag "$sampleId-$replicate" publishDir "$baseDir/output/${task.process}", mode: 'copy' input: set sampleId, deduped, experimentId, biosample, factor, treatment, replicate, controlId from convertReads output: set sampleId, file('*.tagAlign.gz'), file('*.bed{pe,se}.gz'), experimentId, biosample, factor, treatment, replicate, controlId into tagReads script: if (pairedEnd) { """ python3 $baseDir/scripts/convert_reads.py -b $deduped -p """ } else { """ python3 $baseDir/scripts/convert_reads.py -b $deduped """ } } // Calculate Cross-correlation using phantompeaktools process crossReads { tag "$sampleId-$replicate" publishDir "$baseDir/output/${task.process}", mode: 'copy' input: set sampleId, seTagAlign, tagAlign, experimentId, biosample, factor, treatment, replicate, controlId from tagReads output: set sampleId, seTagAlign, tagAlign, file('*.cc.qc'), experimentId, biosample, factor, treatment, replicate, controlId into xcorReads set file('*.cc.qc'), file('*.cc.plot.pdf') into xcorReadsStats script: if (pairedEnd) { """ python3 $baseDir/scripts/xcor.py -t $seTagAlign -p """ } else { """ python3 $baseDir/scripts/xcor.py -t $seTagAlign """ } } // Define channel collecting tagAlign and xcor into design file xcorDesign = xcorReads .map{ sampleId, seTagAlign, tagAlign, xcor, experimentId, biosample, factor, treatment, replicate, controlId -> "$sampleId\t$seTagAlign\t$tagAlign\t$xcor\t$experimentId\t$biosample\t$factor\t$treatment\t$replicate\t$controlId\n"} .collectFile(name:'design_xcor.tsv', seed:"sample_id\tse_tag_align\ttag_align\txcor\texperiment_id\tbiosample\tfactor\ttreatment\treplicate\tcontrol_id\n", storeDir:"$baseDir/output/design") // Make Experiment design files to be read in for downstream analysis process defineExpDesignFiles { publishDir "$baseDir/output/design", mode: 'copy' input: file xcorDesign output: file '*.tsv' into experimentObjs mode flatten script: """ python3 $baseDir/scripts/experiment_design.py -d $xcorDesign """ } // Make Experiment design files to be read in for downstream analysis process poolAndPsuedoReads { tag "${experimentObjs.baseName}" publishDir "$baseDir/output/design", mode: 'copy' input: file experimentObjs output: file '*.tsv' into experimentPoolObjs script: if (pairedEnd) { """ python3 $baseDir/scripts/pool_and_psuedoreplicate.py -d $experimentObjs -c $cutoffRatio -p """ } else { """ python3 $baseDir/scripts/pool_and_psuedoreplicate.py -d $experimentObjs -c $cutoffRatio """ } } // Collect list of experiment design files into a single channel experimentRows = experimentPoolObjs .splitCsv(sep:'\t', header:true) .map { row -> [ row.sample_id, row.tag_align, row.xcor, row.experiment_id, row.biosample, row.factor, row.treatment, row.replicate, row.control_id, row.control_tag_align] } // Call Peaks using MACS process callPeaksMACS { tag "$sampleId-$replicate" publishDir "$baseDir/output/${task.process}", mode: 'copy' input: set sampleId, tagAlign, xcor, experimentId, biosample, factor, treatment, replicate, controlId, controlTagAlign from experimentRows output: set sampleId, file('*.narrowPeak'), file('*.fc_signal.bw'), file('*.pvalue_signal.bw'), experimentId, biosample, factor, treatment, replicate, controlId into experimentPeaks script: if (pairedEnd) { """ python3 $baseDir/scripts/call_peaks_macs.py -t $tagAlign -x $xcor -c $controlTagAlign -s $sampleId -g $genomeSize -z $chromSizes -p """ } else { """ python3 $baseDir/scripts/call_peaks_macs.py -t $tagAlign -x $xcor -c $controlTagAlign -s $sampleId -g $genomeSize -z $chromSizes """ } } // Define channel collecting peaks into design file peaksDesign = experimentPeaks .map{ sampleId, peak, fcSignal, pvalueSignal, experimentId, biosample, factor, treatment, replicate, controlId -> "$sampleId\t$peak\t$fcSignal\t$pvalueSignal\t$experimentId\t$biosample\t$factor\t$treatment\t$replicate\t$controlId\n"} .collectFile(name:'design_peak.tsv', seed:"sample_id\tpeaks\tfc_signal\tpvalue_signal\texperiment_id\tbiosample\tfactor\ttreatment\treplicate\tcontrol_id\n", storeDir:"$baseDir/output/design") // Calculate Consensus Peaks process consensusPeaks { publishDir "$baseDir/output/${task.process}", mode: 'copy' input: file peaksDesign file preDiffDesign output: file '*.replicated.*' into consensusPeaks file '*.rejected.*' into rejectedPeaks file("design_diffPeaks.tsv") into designDiffPeaks script: """ python3 $baseDir/scripts/overlap_peaks.py -d $peaksDesign -f $preDiffDesign """ }