ChIP-seq peak-calling, QC and differential analysis pipeline.

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Introduction

nfcore/chipseq is a bioinformatics analysis pipeline used for Chromatin ImmunopreciPitation sequencing (ChIP-seq) data.

On release, automated continuous integration tests run the pipeline on a full-sized dataset on the AWS cloud infrastructure. The dataset consists of FoxA1 (transcription factor) and EZH2 (histone,mark) IP experiments from Franco et al. 2015 ( GEO: GSE59530 , PMID: 25752574 ) and Popovic et al. 2014 ( GEO: GSE57632 , PMID: 25188243 ), respectively. This ensures that the pipeline runs on AWS, has sensible resource allocation defaults set to run on real-world datasets, and permits the persistent storage of results to benchmark between pipeline releases and other analysis sources. The results obtained from running the full-sized tests can be viewed on the nf-core website .

The pipeline is built using Nextflow , a workflow tool to run tasks across multiple compute infrastructures in a very portable manner. It uses Docker/Singularity containers making installation trivial and results highly reproducible. The Nextflow DSL2 implementation of this pipeline uses one container per process which makes it much easier to maintain and update software dependencies. Where possible, these processes have been submitted to and installed from nf-core/modules in order to make them available to all nf-core pipelines, and to everyone within the Nextflow community!

Online videos

A short talk about the history, current status and functionality on offer in this pipeline was given by Jose Espinosa-Carrasco ( @joseespinosa ) on 26th July 2022 as part of the nf-core/bytesize series.

You can find numerous talks on the nf-core events page from various topics including writing pipelines/modules in Nextflow DSL2, using nf-core tooling, running nf-core pipelines as well as more generic content like contributing to Github. Please check them out!

Pipeline summary

Raw read QC ( FastQC )
Adapter trimming ( Trim Galore! )
Choice of multiple aligners 1.( BWA ) 2.( Chromap ). For paired-end reads only working until mapping steps, see here 3.( Bowtie2 ) 4.( STAR )
Mark duplicates ( picard )
Merge alignments from multiple libraries of the same sample ( picard )
1. Re-mark duplicates ( picard )
2. Filtering to remove:
  - reads mapping to blacklisted regions ( SAMtools , BEDTools )
  - reads that are marked as duplicates ( SAMtools )
  - reads that are not marked as primary alignments ( SAMtools )
  - reads that are unmapped ( SAMtools )
  - reads that map to multiple locations ( SAMtools )
  - reads containing > 4 mismatches ( BAMTools )
  - reads that have an insert size > 2kb ( BAMTools ; paired-end only )
  - reads that map to different chromosomes ( Pysam ; paired-end only )
  - reads that arent in FR orientation ( Pysam ; paired-end only )
  - reads where only one read of the pair fails the above criteria ( Pysam ; paired-end only )
3. Alignment-level QC and estimation of library complexity ( picard , Preseq )
4. Create normalised bigWig files scaled to 1 million mapped reads ( BEDTools , bedGraphToBigWig )
5. Generate gene-body meta-profile from bigWig files ( deepTools )
6. Calculate genome-wide IP enrichment relative to control ( deepTools )
7. Calculate strand cross-correlation peak and ChIP-seq quality measures including NSC and RSC ( phantompeakqualtools )
8. Call broad/narrow peaks ( MACS2 )
9. Annotate peaks relative to gene features ( HOMER )
10. Create consensus peakset across all samples and create tabular file to aid in the filtering of the data ( BEDTools )
11. Count reads in consensus peaks ( featureCounts )
12. PCA and clustering ( R , DESeq2 )
Create IGV session file containing bigWig tracks, peaks and differential sites for data visualisation ( IGV ).
Present QC for raw read, alignment, peak-calling and differential binding results ( MultiQC , R )

Quick Start

Install Nextflow ( >=21.10.3 )
Install any of Docker , Singularity (you can follow this tutorial ), Podman , Shifter or Charliecloud for full pipeline reproducibility (you can use Conda both to install Nextflow itself and also to manage software within pipelines. Please only use it within pipelines as a last resort; see docs ) .
Download the pipeline and test it on a minimal dataset with a single command:
```
nextflow run nf-core/chipseq -profile test,YOURPROFILE --outdir <OUTDIR>
```
Note that some form of configuration will be needed so that Nextflow knows how to fetch the required software. This is usually done in the form of a config profile ( YOURPROFILE in the example command above). You can chain multiple config profiles in a comma-separated string.
- The pipeline comes with config profiles called docker , singularity , podman , shifter , charliecloud and conda which instruct the pipeline to use the named tool for software management. For example, -profile test,docker .
- Please check nf-core/configs to see if a custom config file to run nf-core pipelines already exists for your Institute. If so, you can simply use -profile <institute> in your command. This will enable either docker or singularity and set the appropriate execution settings for your local compute environment.
- If you are using singularity , please use the nf-core download command to download images first, before running the pipeline. Setting the NXF_SINGULARITY_CACHEDIR or singularity.cacheDir Nextflow options enables you to store and re-use the images from a central location for future pipeline runs.
- If you are using conda , it is highly recommended to use the NXF_CONDA_CACHEDIR or conda.cacheDir settings to store the environments in a central location for future pipeline runs.

Start running your own analysis!

nextflow run nf-core/chipseq --input samplesheet.csv --outdir <OUTDIR> --genome GRCh37 -profile <docker/singularity/podman/shifter/charliecloud/conda/institute>

See usage docs for all of the available options when running the pipeline.

Documentation

The nf-core/chipseq pipeline comes with documentation about the pipeline: usage and output .

Credits

These scripts were originally written by Chuan Wang ( @chuan-wang ) and Phil Ewels ( @ewels ) for use at the National Genomics Infrastructure at SciLifeLab in Stockholm, Sweden. The pipeline was re-implemented by Harshil Patel ( @drpatelh ) from Seqera Labs, Spain and converted to Nextflow DSL2 by Jose Espinosa-Carrasco ( @JoseEspinosa ) from The Comparative Bioinformatics Group at The Centre for Genomic Regulation, Spain .

Many thanks to others who have helped out and contributed along the way too, including (but not limited to): @apeltzer , @bc2zb , @crickbabs , @drejom , @houghtos , @KevinMenden , @mashehu , @pditommaso , @Rotholandus , @sofiahaglund , @tiagochst and @winni2k .

Contributions and Support

If you would like to contribute to this pipeline, please see the contributing guidelines .

For further information or help, don't hesitate to get in touch on the Slack #chipseq channel (you can join with this invite ).

Citations

If you use nf-core/chipseq for your analysis, please cite it using the following doi: 10.5281/zenodo.3240506

An extensive list of references for the tools used by the pipeline can be found in the CITATIONS.md file.

You can cite the nf-core publication as follows:

The nf-core framework for community-curated bioinformatics pipelines.

Philip Ewels, Alexander Peltzer, Sven Fillinger, Harshil Patel, Johannes Alneberg, Andreas Wilm, Maxime Ulysse Garcia, Paolo Di Tommaso & Sven Nahnsen.

Nat Biotechnol. 2020 Feb 13. doi: 10.1038/s41587-020-0439-x .

Code Snippets

"""
cut -f2- ${homer_peaks} | awk 'NR==1; NR > 1 {print \$0 | "sort -T '.' -k1,1 -k2,2n"}' | cut -f6- > tmp.txt
paste $boolean_txt tmp.txt > ${prefix}.boolean.annotatePeaks.txt

cat <<-END_VERSIONS > versions.yml
"${task.process}":
    sed: \$(echo \$(sed --version 2>&1) | sed 's/^.*GNU sed) //; s/ .*\$//')
END_VERSIONS
"""

NextFlow From line 19 of local/annotate_boolean_peaks.nf

"""
samtools view \\
    $filter_params \\
    $dup_params \\
    $multimap_params \\
    $blacklist_params \\
    -b $bam \\
    | bamtools filter \\
        -out ${prefix}.bam \\
        -script $config

cat <<-END_VERSIONS > versions.yml
"${task.process}":
    samtools: \$(echo \$(samtools --version 2>&1) | sed 's/^.*samtools //; s/Using.*\$//')
    bamtools: \$(echo \$(bamtools --version 2>&1) | sed 's/^.*bamtools //; s/Part .*\$//')
END_VERSIONS
"""

NextFlow SAMtools BamTools From line 30 of local/bam_filter.nf

"""
samtools sort -n -@ $task.cpus -o ${prefix}.name.sorted.bam -T ${prefix}.name.sorted $bam
bampe_rm_orphan.py ${prefix}.name.sorted.bam ${prefix}.bam $args

cat <<-END_VERSIONS > versions.yml
"${task.process}":
    samtools: \$(echo \$(samtools --version 2>&1) | sed 's/^.*samtools //; s/Using.*\$//')
END_VERSIONS
"""

NextFlow SAMtools From line 24 of local/bam_remove_orphans.nf

"""
ln -s $bam ${prefix}.bam

cat <<-END_VERSIONS > versions.yml
"${task.process}":
    samtools: \$(echo \$(samtools --version 2>&1) | sed 's/^.*samtools //; s/Using.*\$//')
END_VERSIONS
"""

NextFlow From line 34 of local/bam_remove_orphans.nf

"""
SCALE_FACTOR=\$(grep '[0-9] mapped (' $flagstat | awk '{print 1000000/\$1}')
echo \$SCALE_FACTOR > ${prefix}.scale_factor.txt

bedtools \\
    genomecov \\
    -ibam $bam \\
    -bg \\
    -scale \$SCALE_FACTOR \\
    $pe \\
    $extend \\
    | sort -T '.' -k1,1 -k2,2n > ${prefix}.bedGraph

cat <<-END_VERSIONS > versions.yml
"${task.process}":
    bedtools: \$(bedtools --version | sed -e "s/bedtools v//g")
END_VERSIONS
"""

NextFlow BEDTools From line 23 of local/bedtools_genomecov.nf

"""
deseq2_qc.r \\
    --count_file $counts \\
    --outdir ./ \\
    --outprefix $prefix \\
    --cores $task.cpus \\
    $args

sed 's/deseq2_pca/deseq2_pca_${task.index}/g' <$deseq2_pca_header >tmp.txt
sed -i -e 's/DESeq2 /${meta.id} DESeq2 /g' tmp.txt
cat tmp.txt ${prefix}.pca.vals.txt > ${prefix}.pca.vals_mqc.tsv

sed 's/deseq2_clustering/deseq2_clustering_${task.index}/g' <$deseq2_clustering_header >tmp.txt
sed -i -e 's/DESeq2 /${meta.id} DESeq2 /g' tmp.txt
cat tmp.txt ${prefix}.sample.dists.txt > ${prefix}.sample.dists_mqc.tsv

cat <<-END_VERSIONS > versions.yml
"${task.process}":
    r-base: \$(echo \$(R --version 2>&1) | sed 's/^.*R version //; s/ .*\$//')
    bioconductor-deseq2: \$(Rscript -e "library(DESeq2); cat(as.character(packageVersion('DESeq2')))")
END_VERSIONS
"""

NextFlow DESeq2 From line 33 of local/deseq2_qc.nf

"""
READS_IN_PEAKS=\$(intersectBed -a $bam -b $peak $args | awk -F '\t' '{sum += \$NF} END {print sum}')
samtools flagstat $bam > ${bam}.flagstat
grep 'mapped (' ${bam}.flagstat | grep -v "primary" | awk -v a="\$READS_IN_PEAKS" -v OFS='\t' '{print "${prefix}", a/\$1}' > ${prefix}.FRiP.txt

cat <<-END_VERSIONS > versions.yml
"${task.process}":
    bedtools: \$(bedtools --version | sed -e "s/bedtools v//g")
    samtools: \$(echo \$(samtools --version 2>&1) | sed 's/^.*samtools //; s/Using.*\$//')
END_VERSIONS
"""

NextFlow SAMtools BEDTools From line 20 of local/frip_score.nf

"""
sortBed -i $blacklist -g $sizes | complementBed -i stdin -g $sizes > $file_out

cat <<-END_VERSIONS > versions.yml
"${task.process}":
    bedtools: \$(bedtools --version | sed -e "s/bedtools v//g")
END_VERSIONS
"""

NextFlow BEDTools From line 23 of local/genome_blacklist_regions.nf

"""
awk '{print \$1, '0' , \$2}' OFS='\t' $sizes > $file_out

cat <<-END_VERSIONS > versions.yml
"${task.process}":
    bedtools: \$(bedtools --version | sed -e "s/bedtools v//g")
END_VERSIONS
"""

NextFlow BEDTools From line 32 of local/genome_blacklist_regions.nf

"""
gtf2bed \\
    $gtf \\
    > ${gtf.baseName}.bed

cat <<-END_VERSIONS > versions.yml
"${task.process}":
    perl: \$(echo \$(perl --version 2>&1) | sed 's/.*v\\(.*\\)) built.*/\\1/')
END_VERSIONS
"""

NextFlow GFFutils From line 18 of local/gtf2bed.nf

"""
find * -type l -name "*.bigWig" -exec echo -e ""{}"\\t0,0,178" \\; > bigwig.igv.txt
find * -type l -name "*Peak" -exec echo -e ""{}"\\t0,0,178" \\; > peaks.igv.txt
# Avoid error when consensus not produced
find * -type l -name "*.bed" -exec echo -e ""{}"\\t0,0,178" \\; | { grep "^$consensus_dir" || test \$? = 1; } > consensus.igv.txt

touch replace_paths.txt
if [ -d "mappings" ]; then
    cat mappings/* > replace_paths.txt
fi

cat *.igv.txt > igv_files_orig.txt
igv_files_to_session.py igv_session.xml igv_files_orig.txt replace_paths.txt ../../genome/${fasta.getName()} --path_prefix '../../'

cat <<-END_VERSIONS > versions.yml
"${task.process}":
    python: \$(python --version | sed 's/Python //g')
END_VERSIONS
"""

NextFlow Consensus From line 27 of local/igv.nf

"""
sort -T '.' -k1,1 -k2,2n ${peaks.collect{it.toString()}.sort().join(' ')} \\
    | mergeBed -c $mergecols -o $collapsecols > ${prefix}.txt

macs2_merged_expand.py \\
    ${prefix}.txt \\
    ${peaks.collect{it.toString()}.sort().join(',').replaceAll("_peaks.${peak_type}","")} \\
    ${prefix}.boolean.txt \\
    --min_replicates $params.min_reps_consensus \\
    $expandparam

awk -v FS='\t' -v OFS='\t' 'FNR > 1 { print \$1, \$2, \$3, \$4, "0", "+" }' ${prefix}.boolean.txt > ${prefix}.bed

echo -e "GeneID\tChr\tStart\tEnd\tStrand" > ${prefix}.saf
awk -v FS='\t' -v OFS='\t' 'FNR > 1 { print \$4, \$1, \$2, \$3,  "+" }' ${prefix}.boolean.txt >> ${prefix}.saf

plot_peak_intersect.r -i ${prefix}.boolean.intersect.txt -o ${prefix}.boolean.intersect.plot.pdf

echo "${prefix}.bed\t${meta.id}/${prefix}.bed" > ${prefix}.antibody.txt

cat <<-END_VERSIONS > versions.yml
"${task.process}":
    python: \$(python --version | sed 's/Python //g')
    r-base: \$(echo \$(R --version 2>&1) | sed 's/^.*R version //; s/ .*\$//')
END_VERSIONS
"""

NextFlow From line 34 of local/macs2_consensus.nf

"""
cat $peak | wc -l | awk -v OFS='\t' '{ print "${prefix}", \$1 }' | cat $peak_count_header - > ${prefix}.peak_count_mqc.tsv
cat $frip_score_header $frip > ${prefix}.FRiP_mqc.tsv

cat <<-END_VERSIONS > versions.yml
"${task.process}":
    sed: \$(echo \$(sed --version 2>&1) | sed 's/^.*GNU sed) //; s/ .*\$//')
END_VERSIONS
"""

NextFlow From line 19 of local/multiqc_custom_peaks.nf

"""
cp $correlation_header ${prefix}.spp_correlation_mqc.tsv
Rscript --max-ppsize=500000 -e "load('$rdata'); write.table(crosscorr\\\$cross.correlation, file=\\"${prefix}.spp_correlation_mqc.tsv\\", sep=",", quote=FALSE, row.names=FALSE, col.names=FALSE,append=TRUE)"

awk -v OFS='\t' '{print "${meta.id}", \$9}'  $spp | cat $nsc_header - > ${prefix}.spp_nsc_mqc.tsv
awk -v OFS='\t' '{print "${meta.id}", \$10}' $spp | cat $rsc_header - > ${prefix}.spp_rsc_mqc.tsv

cat <<-END_VERSIONS > versions.yml
"${task.process}":
    r-base: \$(echo \$(R --version 2>&1) | sed 's/^.*R version //; s/ .*\$//')
END_VERSIONS
"""

NextFlow From line 21 of local/multiqc_custom_phantompeakqualtools.nf

"""
multiqc \\
    -f \\
    $args \\
    $custom_config \\
    .

cat <<-END_VERSIONS > versions.yml
"${task.process}":
    multiqc: \$( multiqc --version | sed -e "s/multiqc, version //g" )
END_VERSIONS
"""

NextFlow MultiQC From line 60 of local/multiqc.nf

"""
plot_homer_annotatepeaks.r \\
    -i ${annos.join(',')} \\
    -s ${annos.join(',').replaceAll("${suffix}","")} \\
    -p $prefix \\
    $args

find ./ -type f -name "*summary.txt" -exec cat {} \\; | cat $mqc_header - > ${prefix}.summary_mqc.tsv

cat <<-END_VERSIONS > versions.yml
"${task.process}":
    r-base: \$(echo \$(R --version 2>&1) | sed 's/^.*R version //; s/ .*\$//')
END_VERSIONS
"""

NextFlow From line 23 of local/plot_homer_annotatepeaks.nf

"""
plot_macs2_qc.r \\
    -i ${peaks.join(',')} \\
    -s ${peaks.join(',').replaceAll("_peaks.${peak_type}","")} \\
    $args

cat <<-END_VERSIONS > versions.yml
"${task.process}":
    r-base: \$(echo \$(R --version 2>&1) | sed 's/^.*R version //; s/ .*\$//')
END_VERSIONS
"""

NextFlow From line 20 of local/plot_macs2_qc.nf

"""
check_samplesheet.py \\
    $samplesheet \\
    samplesheet.valid.csv

cat <<-END_VERSIONS > versions.yml
"${task.process}":
    python: \$(python --version | sed 's/Python //g')
END_VERSIONS
"""

NextFlow From line 17 of local/samplesheet_check.nf

"""
STAR \\
    --genomeDir $index \\
    --readFilesIn $reads  \\
    --runThreadN $task.cpus \\
    --outFileNamePrefix $prefix. \\
    $out_sam_type \\
    $seq_center \\
    $args
$mv_unsorted_bam
if [ -f ${prefix}.Unmapped.out.mate1 ]; then
    mv ${prefix}.Unmapped.out.mate1 ${prefix}.unmapped_1.fastq
    gzip ${prefix}.unmapped_1.fastq
fi
if [ -f ${prefix}.Unmapped.out.mate2 ]; then
    mv ${prefix}.Unmapped.out.mate2 ${prefix}.unmapped_2.fastq
    gzip ${prefix}.unmapped_2.fastq
fi
cat <<-END_VERSIONS > versions.yml
"${task.process}":
    star: \$(STAR --version | sed -e "s/STAR_//g")
END_VERSIONS
"""

NextFlow STAR From line 34 of local/star_align.nf

"""
mkdir star
STAR \\
    --runMode genomeGenerate \\
    --genomeDir star/ \\
    --genomeFastaFiles $fasta \\
    --sjdbGTFfile $gtf \\
    --runThreadN $task.cpus \\
    $memory \\
    ${args.join(' ')}
cat <<-END_VERSIONS > versions.yml
"${task.process}":
    star: \$(STAR --version | sed -e "s/STAR_//g")
END_VERSIONS
"""

NextFlow STAR From line 23 of local/star_genomegenerate.nf

"""
samtools faidx $fasta
NUM_BASES=`gawk '{sum = sum + \$2}END{if ((log(sum)/log(2))/2 - 1 > 14) {printf "%.0f", 14} else {printf "%.0f", (log(sum)/log(2))/2 - 1}}' ${fasta}.fai`
mkdir star
STAR \\
    --runMode genomeGenerate \\
    --genomeDir star/ \\
    --genomeFastaFiles $fasta \\
    --sjdbGTFfile $gtf \\
    --runThreadN $task.cpus \\
    --genomeSAindexNbases \$NUM_BASES \\
    $memory \\
    ${args.join(' ')}
cat <<-END_VERSIONS > versions.yml
"${task.process}":
    star: \$(STAR --version | sed -e "s/STAR_//g")
END_VERSIONS
"""

NextFlow SAMtools STAR From line 39 of local/star_genomegenerate.nf

"""
INDEX=`find -L ./ -name "*.rev.1.bt2" | sed "s/.rev.1.bt2//"`
[ -z "\$INDEX" ] && INDEX=`find -L ./ -name "*.rev.1.bt2l" | sed "s/.rev.1.bt2l//"`
[ -z "\$INDEX" ] && echo "Bowtie2 index files not found" 1>&2 && exit 1

bowtie2 \\
    -x \$INDEX \\
    $reads_args \\
    --threads $task.cpus \\
    $unaligned \\
    $args \\
    2> ${prefix}.bowtie2.log \\
    | samtools $samtools_command $args2 --threads $task.cpus -o ${prefix}.bam -

if [ -f ${prefix}.unmapped.fastq.1.gz ]; then
    mv ${prefix}.unmapped.fastq.1.gz ${prefix}.unmapped_1.fastq.gz
fi

if [ -f ${prefix}.unmapped.fastq.2.gz ]; then
    mv ${prefix}.unmapped.fastq.2.gz ${prefix}.unmapped_2.fastq.gz
fi

cat <<-END_VERSIONS > versions.yml
"${task.process}":
    bowtie2: \$(echo \$(bowtie2 --version 2>&1) | sed 's/^.*bowtie2-align-s version //; s/ .*\$//')
    samtools: \$(echo \$(samtools --version 2>&1) | sed 's/^.*samtools //; s/Using.*\$//')
    pigz: \$( pigz --version 2>&1 | sed 's/pigz //g' )
END_VERSIONS
"""

NextFlow SAMtools Bowtie 2 From line 42 of align/main.nf

"""
mkdir bowtie2
bowtie2-build $args --threads $task.cpus $fasta bowtie2/${fasta.baseName}
cat <<-END_VERSIONS > versions.yml
"${task.process}":
    bowtie2: \$(echo \$(bowtie2 --version 2>&1) | sed 's/^.*bowtie2-align-s version //; s/ .*\$//')
END_VERSIONS
"""

NextFlow Bowtie 2 From line 22 of build/main.nf

"""
mkdir bwa
bwa \\
    index \\
    $args \\
    -p bwa/${fasta.baseName} \\
    $fasta

cat <<-END_VERSIONS > versions.yml
"${task.process}":
    bwa: \$(echo \$(bwa 2>&1) | sed 's/^.*Version: //; s/Contact:.*\$//')
END_VERSIONS
"""

NextFlow BWA From line 22 of index/main.nf

"""
INDEX=`find -L ./ -name "*.amb" | sed 's/.amb//'`

bwa mem \\
    $args \\
    -t $task.cpus \\
    \$INDEX \\
    $reads \\
    | samtools $samtools_command $args2 --threads $task.cpus -o ${prefix}.bam -

cat <<-END_VERSIONS > versions.yml
"${task.process}":
    bwa: \$(echo \$(bwa 2>&1) | sed 's/^.*Version: //; s/Contact:.*\$//')
    samtools: \$(echo \$(samtools --version 2>&1) | sed 's/^.*samtools //; s/Using.*\$//')
END_VERSIONS
"""

NextFlow SAMtools BWA From line 27 of mem/main.nf

    """
}
if (meta.single_end) {
    """

NextFlow From line 55 of chromap/main.nf

    """
} else {
    """

NextFlow From line 74 of chromap/main.nf

"""

NextFlow From line 93 of chromap/main.nf

"""
chromap \\
    -i \\
    $args \\
    -t $task.cpus \\
    -r $fasta \\
    -o ${prefix}.index

cat <<-END_VERSIONS > versions.yml
"${task.process}":
    chromap: \$(echo \$(chromap --version 2>&1))
END_VERSIONS
"""

NextFlow chromap From line 23 of index/main.nf

"""
samtools faidx $fasta
cut -f 1,2 ${fasta}.fai > ${fasta}.sizes

cat <<-END_VERSIONS > versions.yml
"${task.process}":
    custom: \$(echo \$(samtools --version 2>&1) | sed 's/^.*samtools //; s/Using.*\$//')
END_VERSIONS
"""

NextFlow SAMtools From line 23 of getchromsizes/main.nf

"""
computeMatrix \\
    $args \\
    --regionsFileName $bed \\
    --scoreFileName $bigwig \\
    --outFileName ${prefix}.computeMatrix.mat.gz \\
    --outFileNameMatrix ${prefix}.computeMatrix.vals.mat.tab \\
    --numberOfProcessors $task.cpus

cat <<-END_VERSIONS > versions.yml
"${task.process}":
    deeptools: \$(computeMatrix --version | sed -e "s/computeMatrix //g")
END_VERSIONS
"""

NextFlow DeepTools From line 25 of computematrix/main.nf

"""
plotFingerprint \\
    $args \\
    $extend \\
    --bamfiles ${bams.join(' ')} \\
    --plotFile ${prefix}.plotFingerprint.pdf \\
    --outRawCounts ${prefix}.plotFingerprint.raw.txt \\
    --outQualityMetrics ${prefix}.plotFingerprint.qcmetrics.txt \\
    --numberOfProcessors $task.cpus

cat <<-END_VERSIONS > versions.yml
"${task.process}":
    deeptools: \$(plotFingerprint --version | sed -e "s/plotFingerprint //g")
END_VERSIONS
"""

NextFlow DeepTools From line 26 of plotfingerprint/main.nf

"""
plotHeatmap \\
    $args \\
    --matrixFile $matrix \\
    --outFileName ${prefix}.plotHeatmap.pdf \\
    --outFileNameMatrix ${prefix}.plotHeatmap.mat.tab

cat <<-END_VERSIONS > versions.yml
"${task.process}":
    deeptools: \$(plotHeatmap --version | sed -e "s/plotHeatmap //g")
END_VERSIONS
"""

NextFlow DeepTools From line 24 of plotheatmap/main.nf

"""
plotProfile \\
    $args \\
    --matrixFile $matrix \\
    --outFileName ${prefix}.plotProfile.pdf \\
    --outFileNameData ${prefix}.plotProfile.tab

cat <<-END_VERSIONS > versions.yml
"${task.process}":
    deeptools: \$(plotProfile --version | sed -e "s/plotProfile //g")
END_VERSIONS
"""

NextFlow DeepTools From line 24 of plotprofile/main.nf

"""
[ ! -f  ${prefix}.fastq.gz ] && ln -s $reads ${prefix}.fastq.gz
fastqc $args --threads $task.cpus ${prefix}.fastq.gz

cat <<-END_VERSIONS > versions.yml
"${task.process}":
    fastqc: \$( fastqc --version | sed -e "s/FastQC v//g" )
END_VERSIONS
"""

NextFlow FastQC From line 26 of fastqc/main.nf

"""
[ ! -f  ${prefix}_1.fastq.gz ] && ln -s ${reads[0]} ${prefix}_1.fastq.gz
[ ! -f  ${prefix}_2.fastq.gz ] && ln -s ${reads[1]} ${prefix}_2.fastq.gz
fastqc $args --threads $task.cpus ${prefix}_1.fastq.gz ${prefix}_2.fastq.gz

cat <<-END_VERSIONS > versions.yml
"${task.process}":
    fastqc: \$( fastqc --version | sed -e "s/FastQC v//g" )
END_VERSIONS
"""

NextFlow FastQC From line 36 of fastqc/main.nf

"""
touch ${prefix}.html
touch ${prefix}.zip

cat <<-END_VERSIONS > versions.yml
"${task.process}":
    fastqc: \$( fastqc --version | sed -e "s/FastQC v//g" )
END_VERSIONS
"""

NextFlow FastQC From line 50 of fastqc/main.nf

"""
gffread \\
    $gff \\
    $args \\
    -o ${prefix}.gtf
cat <<-END_VERSIONS > versions.yml
"${task.process}":
    gffread: \$(gffread --version 2>&1)
END_VERSIONS
"""

NextFlow gffread From line 23 of gffread/main.nf

"""
gunzip \\
    -f \\
    $args \\
    $archive

cat <<-END_VERSIONS > versions.yml
"${task.process}":
    gunzip: \$(echo \$(gunzip --version 2>&1) | sed 's/^.*(gzip) //; s/ Copyright.*\$//')
END_VERSIONS
"""

NextFlow From line 23 of gunzip/main.nf

"""
touch $gunzip
cat <<-END_VERSIONS > versions.yml
"${task.process}":
    gunzip: \$(echo \$(gunzip --version 2>&1) | sed 's/^.*(gzip) //; s/ Copyright.*\$//')
END_VERSIONS
"""

NextFlow From line 37 of gunzip/main.nf

"""
annotatePeaks.pl \\
    $peak \\
    $fasta \\
    $args \\
    -gtf $gtf \\
    -cpu $task.cpus \\
    > ${prefix}.annotatePeaks.txt

cat <<-END_VERSIONS > versions.yml
"${task.process}":
    homer: $VERSION
END_VERSIONS
"""

NextFlow homer From line 27 of annotatepeaks/main.nf

"""
unique-kmers.py \\
    -k $kmer_size \\
    -R report.txt \\
    $args \\
    $fasta

grep ^number report.txt | sed 's/^.*:.[[:blank:]]//g' > kmers.txt

cat <<-END_VERSIONS > versions.yml
"${task.process}":
    khmer: \$( unique-kmers.py --version 2>&1 | grep ^khmer | sed 's/^khmer //;s/ .*\$//' )
END_VERSIONS
"""

NextFlow khmer From line 24 of uniquekmers/main.nf

"""
macs2 \\
    callpeak \\
    ${args_list.join(' ')} \\
    --gsize $macs2_gsize \\
    --format $format \\
    --name $prefix \\
    --treatment $ipbam \\
    $control

cat <<-END_VERSIONS > versions.yml
"${task.process}":
    macs2: \$(macs2 --version | sed -e "s/macs2 //g")
END_VERSIONS
"""

NextFlow macs2 From line 38 of callpeak/main.nf

"""
RUN_SPP=`which run_spp.R`
Rscript $args -e "library(caTools); source(\\"\$RUN_SPP\\")" -c="$bam" -savp="${prefix}.spp.pdf" -savd="${prefix}.spp.Rdata" -out="${prefix}.spp.out" $args2

cat <<-END_VERSIONS > versions.yml
"${task.process}":
    phantompeakqualtools: $VERSION
END_VERSIONS
"""

NextFlow From line 28 of phantompeakqualtools/main.nf

"""
picard \\
    -Xmx${avail_mem}g \\
    CollectMultipleMetrics \\
    $args \\
    --INPUT $bam \\
    --OUTPUT ${prefix}.CollectMultipleMetrics \\
    $reference

cat <<-END_VERSIONS > versions.yml
"${task.process}":
    picard: \$(picard CollectMultipleMetrics --version 2>&1 | grep -o 'Version.*' | cut -f2- -d:)
END_VERSIONS
"""

NextFlow Picard From line 33 of collectmultiplemetrics/main.nf

"""
touch ${prefix}.CollectMultipleMetrics.alignment_summary_metrics
touch ${prefix}.CollectMultipleMetrics.insert_size_metrics
touch ${prefix}.CollectMultipleMetrics.quality_distribution.pdf
touch ${prefix}.CollectMultipleMetrics.base_distribution_by_cycle_metrics
touch ${prefix}.CollectMultipleMetrics.quality_by_cycle_metrics
touch ${prefix}.CollectMultipleMetrics.read_length_histogram.pdf
touch ${prefix}.CollectMultipleMetrics.base_distribution_by_cycle.pdf
touch ${prefix}.CollectMultipleMetrics.quality_by_cycle.pdf
touch ${prefix}.CollectMultipleMetrics.insert_size_histogram.pdf
touch ${prefix}.CollectMultipleMetrics.quality_distribution_metrics

cat <<-END_VERSIONS > versions.yml
"${task.process}":
    picard: \$(echo \$(picard CollectMultipleMetrics --version 2>&1) | grep -o 'Version:.*' | cut -f2- -d:)
END_VERSIONS
"""

NextFlow From line 50 of collectmultiplemetrics/main.nf

"""
picard \\
    -Xmx${avail_mem}g \\
    MarkDuplicates \\
    $args \\
    --INPUT $bam \\
    --OUTPUT ${prefix}.bam \\
    --METRICS_FILE ${prefix}.MarkDuplicates.metrics.txt

cat <<-END_VERSIONS > versions.yml
"${task.process}":
    picard: \$(echo \$(picard MarkDuplicates --version 2>&1) | grep -o 'Version:.*' | cut -f2- -d:)
END_VERSIONS
"""

NextFlow Picard From line 31 of markduplicates/main.nf

"""
touch ${prefix}.bam
touch ${prefix}.bam.bai
touch ${prefix}.MarkDuplicates.metrics.txt

cat <<-END_VERSIONS > versions.yml
"${task.process}":
    picard: \$(echo \$(picard MarkDuplicates --version 2>&1) | grep -o 'Version:.*' | cut -f2- -d:)
END_VERSIONS
"""

NextFlow From line 48 of markduplicates/main.nf

"""
picard \\
    -Xmx${avail_mem}g \\
    MergeSamFiles \\
    $args \\
    ${'--INPUT '+bam_files.join(' --INPUT ')} \\
    --OUTPUT ${prefix}.bam
cat <<-END_VERSIONS > versions.yml
"${task.process}":
    picard: \$( echo \$(picard MergeSamFiles --version 2>&1) | grep -o 'Version:.*' | cut -f2- -d:)
END_VERSIONS
"""

NextFlow Picard From line 31 of mergesamfiles/main.nf

"""
ln -s ${bam_files[0]} ${prefix}.bam
cat <<-END_VERSIONS > versions.yml
"${task.process}":
    picard: \$( echo \$(picard MergeSamFiles --version 2>&1) | grep -o 'Version:.*' | cut -f2- -d:)
END_VERSIONS
"""

NextFlow From line 44 of mergesamfiles/main.nf

"""
preseq \\
    lc_extrap \\
    $args \\
    $paired_end \\
    -output ${prefix}.lc_extrap.txt \\
    $bam
cp .command.err ${prefix}.command.log

cat <<-END_VERSIONS > versions.yml
"${task.process}":
    preseq: \$(echo \$(preseq 2>&1) | sed 's/^.*Version: //; s/Usage:.*\$//')
END_VERSIONS
"""

NextFlow preseq From line 26 of lcextrap/main.nf

"""
samtools \\
    flagstat \\
    --threads ${task.cpus-1} \\
    $bam \\
    > ${prefix}.flagstat

cat <<-END_VERSIONS > versions.yml
"${task.process}":
    samtools: \$(echo \$(samtools --version 2>&1) | sed 's/^.*samtools //; s/Using.*\$//')
END_VERSIONS
"""

NextFlow SAMtools From line 23 of flagstat/main.nf

"""
samtools \\
    idxstats \\
    $bam \\
    > ${prefix}.idxstats

cat <<-END_VERSIONS > versions.yml
"${task.process}":
    samtools: \$(echo \$(samtools --version 2>&1) | sed 's/^.*samtools //; s/Using.*\$//')
END_VERSIONS
"""

NextFlow SAMtools From line 24 of idxstats/main.nf

"""
samtools \\
    index \\
    -@ ${task.cpus-1} \\
    $args \\
    $input

cat <<-END_VERSIONS > versions.yml
"${task.process}":
    samtools: \$(echo \$(samtools --version 2>&1) | sed 's/^.*samtools //; s/Using.*\$//')
END_VERSIONS
"""

NextFlow SAMtools From line 24 of index/main.nf

"""
touch ${input}.bai
touch ${input}.crai
touch ${input}.csi

cat <<-END_VERSIONS > versions.yml
"${task.process}":
    samtools: \$(echo \$(samtools --version 2>&1) | sed 's/^.*samtools //; s/Using.*\$//')
END_VERSIONS
"""

NextFlow From line 38 of index/main.nf

"""
samtools sort $args -@ $task.cpus -o ${prefix}.bam -T $prefix $bam
cat <<-END_VERSIONS > versions.yml
"${task.process}":
    samtools: \$(echo \$(samtools --version 2>&1) | sed 's/^.*samtools //; s/Using.*\$//')
END_VERSIONS
"""

NextFlow SAMtools From line 24 of sort/main.nf

"""
touch ${prefix}.bam

cat <<-END_VERSIONS > versions.yml
"${task.process}":
    samtools: \$(echo \$(samtools --version 2>&1) | sed 's/^.*samtools //; s/Using.*\$//')
END_VERSIONS
"""

NextFlow From line 34 of sort/main.nf

"""
samtools \\
    stats \\
    --threads ${task.cpus-1} \\
    ${reference} \\
    ${input} \\
    > ${prefix}.stats

cat <<-END_VERSIONS > versions.yml
"${task.process}":
    samtools: \$(echo \$(samtools --version 2>&1) | sed 's/^.*samtools //; s/Using.*\$//')
END_VERSIONS
"""

NextFlow SAMtools From line 25 of stats/main.nf

"""
touch ${prefix}.stats

cat <<-END_VERSIONS > versions.yml
"${task.process}":
    samtools: \$(echo \$(samtools --version 2>&1) | sed 's/^.*samtools //; s/Using.*\$//')
END_VERSIONS
"""

NextFlow From line 41 of stats/main.nf

"""
featureCounts \\
    $args \\
    $paired_end \\
    -T $task.cpus \\
    -a $annotation \\
    -s $strandedness \\
    -o ${prefix}.featureCounts.txt \\
    ${bams.join(' ')}

cat <<-END_VERSIONS > versions.yml
"${task.process}":
    subread: \$( echo \$(featureCounts -v 2>&1) | sed -e "s/featureCounts v//g")
END_VERSIONS
"""

NextFlow FeatureCounts From line 32 of featurecounts/main.nf

"""
[ ! -f  ${prefix}.fastq.gz ] && ln -s $reads ${prefix}.fastq.gz
trim_galore \\
    $args \\
    --cores $cores \\
    --gzip \\
    $c_r1 \\
    $tpc_r1 \\
    ${prefix}.fastq.gz

cat <<-END_VERSIONS > versions.yml
"${task.process}":
    trimgalore: \$(echo \$(trim_galore --version 2>&1) | sed 's/^.*version //; s/Last.*\$//')
    cutadapt: \$(cutadapt --version)
END_VERSIONS
"""

NextFlow Trim_Galore From line 47 of trimgalore/main.nf

"""
[ ! -f  ${prefix}_1.fastq.gz ] && ln -s ${reads[0]} ${prefix}_1.fastq.gz
[ ! -f  ${prefix}_2.fastq.gz ] && ln -s ${reads[1]} ${prefix}_2.fastq.gz
trim_galore \\
    $args \\
    --cores $cores \\
    --paired \\
    --gzip \\
    $c_r1 \\
    $c_r2 \\
    $tpc_r1 \\
    $tpc_r2 \\
    ${prefix}_1.fastq.gz \\
    ${prefix}_2.fastq.gz

cat <<-END_VERSIONS > versions.yml
"${task.process}":
    trimgalore: \$(echo \$(trim_galore --version 2>&1) | sed 's/^.*version //; s/Last.*\$//')
    cutadapt: \$(cutadapt --version)
END_VERSIONS
"""

NextFlow Trim_Galore From line 64 of trimgalore/main.nf

"""
bedGraphToBigWig \\
    $bedgraph \\
    $sizes \\
    ${prefix}.bigWig

cat <<-END_VERSIONS > versions.yml
"${task.process}":
    ucsc: $VERSION
END_VERSIONS
"""

NextFlow bedGraphToBigWig From line 26 of bedgraphtobigwig/main.nf

"""
mkdir output

tar \\
    -C output --strip-components 1 \\
    -xzvf \\
    $args \\
    $archive \\
    $args2

mv output ${untar}

cat <<-END_VERSIONS > versions.yml
"${task.process}":
    untar: \$(echo \$(tar --version 2>&1) | sed 's/^.*(GNU tar) //; s/ Copyright.*\$//')
END_VERSIONS
"""

NextFlow From line 25 of untar/main.nf

"""
touch $untar

cat <<-END_VERSIONS > versions.yml
"${task.process}":
    untar: \$(echo \$(tar --version 2>&1) | sed 's/^.*(GNU tar) //; s/ Copyright.*\$//')
END_VERSIONS
"""