ATAC-seq peak-calling, QC and differential analysis pipeline

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Introduction

nfcore/atacseq is a bioinformatics analysis pipeline used for ATAC-seq data.

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!

On release, automated continuous integration tests run the pipeline on a full-sized dataset on the AWS cloud infrastructure. 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 the full-sized test can be viewed on the nf-core website .

Pipeline summary

nf-core/atacseq metro map

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 mitochondrial DNA ( SAMtools )
  - 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 are soft-clipped ( 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 enrichment (optionally relative to control) ( deepTools )
7. Call broad/narrow peaks ( MACS2 )
8. Annotate peaks relative to gene features ( HOMER )
9. Create consensus peakset across all samples and create tabular file to aid in the filtering of the data ( BEDTools )
10. Count reads in consensus peaks ( featureCounts )
11. Differential accessibility analysis, PCA and clustering ( R , DESeq2 )
12. Generate ATAC-seq specific QC html report ( ataqv )
Merge filtered alignments across replicates ( picard )
1. Re-mark duplicates ( picard )
2. Remove duplicate reads ( SAMtools )
3. Create normalised bigWig files scaled to 1 million mapped reads ( BEDTools , bedGraphToBigWig )
4. Call broad/narrow peaks ( MACS2 )
5. Annotate peaks relative to gene features ( HOMER )
6. Create consensus peakset across all samples and create tabular file to aid in the filtering of the data ( BEDTools )
7. Count reads in consensus peaks relative to merged library-level alignments ( featureCounts )
8. Differential accessibility analysis, 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 accessibility results ( ataqv , MultiQC , R )

Usage

Note If you are new to Nextflow and nf-core, please refer to this page on how to set-up Nextflow. Make sure to test your setup with -profile test before running the workflow on actual data.

To run on your data, prepare a tab-separated samplesheet with your input data. Please follow the documentation on samplesheets for more details. An example samplesheet for running the pipeline looks as follows:

sample,fastq_1,fastq_2,replicate
CONTROL,AEG588A1_S1_L002_R1_001.fastq.gz,AEG588A1_S1_L002_R2_001.fastq.gz,1
CONTROL,AEG588A1_S1_L003_R1_001.fastq.gz,AEG588A1_S1_L003_R2_001.fastq.gz,2
CONTROL,AEG588A1_S1_L004_R1_001.fastq.gz,AEG588A1_S1_L004_R2_001.fastq.gz,3

Now, you can run the pipeline using:

nextflow run nf-core/atacseq --input samplesheet.csv --outdir <OUTDIR> --genome GRCh37 --read_length <50|100|150|200> -profile <docker/singularity/podman/shifter/charliecloud/conda/institute>

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

Warning: Please provide pipeline parameters via the CLI or Nextflow -params-file option. Custom config files including those provided by the -c Nextflow option can be used to provide any configuration except for parameters ; see docs .

For more details and further functionality, please refer to the usage documentation and the parameter documentation .

Pipeline output

To see the results of an example test run with a full size dataset refer to the results tab on the nf-core website pipeline page. For more details about the output files and reports, please refer to the output documentation .

Credits

The pipeline was originally written by Harshil Patel ( @drpatelh ) from Seqera Labs, Spain and converted to Nextflow DSL2 by Björn Langer ( @bjlang ) and Jose Espinosa-Carrasco ( @JoseEspinosa ) from The Comparative Bioinformatics Group at The Centre for Genomic Regulation, Spain under the umbrella of the BovReg project .

Many thanks to others who have helped out and contributed along the way too, including (but not limited to): @ewels , @apeltzer , @crickbabs , drewjbeh , @houghtos , @jinmingda , @ktrns , @MaxUlysse , @mashehu , @micans , @pditommaso and @sven1103 .

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 #atacseq channel (you can join with this invite ).

Citations

If you use nf-core/atacseq for your analysis, please cite it using the following doi: 10.5281/zenodo.2634132

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

"""
bampe_rm_orphan.py \\
    $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 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 36 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 \\
    $args \\
> tmp.bg

bedtools sort -i tmp.bg > ${prefix}.bedGraph

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

NextFlow BEDTools From line 25 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 35 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 23 of local/frip_score.nf

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

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

NextFlow BEDTools From line 27 of local/genome_blacklist_regions.nf

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

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

NextFlow BEDTools From line 36 of local/genome_blacklist_regions.nf

"""
get_autosomes.py \\
    $fai \\
    ${fai.baseName}.autosomes.txt

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

NextFlow From line 20 of local/get_autosomes.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 21 of local/gtf2bed.nf

"""
find * -type l -name "*.bigWig" -exec echo -e ""{}"\\t0,0,178" \\; | { grep "^$bigwig_library_publish_dir" || test \$? = 1; } > mLb_bigwig.igv.txt
find * -type l -name "*Peak" -exec echo -e ""{}"\\t0,0,178" \\; | { grep "^$peak_library_publish_dir" || test \$? = 1; } > mLb_peaks.igv.txt
find * -type l -name "*.bed" -exec echo -e ""{}"\\t0,0,0" \\; | { grep "^$consensus_library_publish_dir" || test \$? = 1; } > mLb_bed.igv.txt
find * -type l -name "*.bigWig" -exec echo -e ""{}"\\t0,0,178" \\; | { grep "^$bigwig_replicate_publish_dir" || test \$? = 1; } > mRp_bigwig.igv.txt
find * -type l -name "*Peak" -exec echo -e ""{}"\\t0,0,178" \\; | { grep "^$peak_replicate_publish_dir" || test \$? = 1; } > mRp_peaks.igv.txt
find * -type l -name "*.bed" -exec echo -e ""{}"\\t0,0,0" \\; | { grep "^$consensus_replicate_publish_dir" || test \$? = 1; } > mRp_bed.igv.txt

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

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

NextFlow From line 36 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 \\
    $args \\
    $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

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 32 of local/macs2_consensus.nf

"""
cat $peak | wc -l | awk -v OFS='\t' '{ print "${prefix}", \$1 }' | cat $peak_count_header - > ${prefix}.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 24 of local/multiqc_custom_peaks.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 70 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 26 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 24 of local/plot_macs2_qc.nf

"""
check_samplesheet.py \\
    $samplesheet \\
    $args

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

NextFlow From line 22 of local/samplesheet_check.nf

"""
STAR \\
    --genomeDir $index \\
    --readFilesIn $reads  \\
    --runThreadN $task.cpus \\
    --outFileNamePrefix $prefix. \\
    $out_sam_type \\
    $seq_center_tag \\
    $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 38 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 26 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 42 of local/star_genomegenerate.nf

"""
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

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/tss_extract.nf

"""
ataqv \\
    $args \\
    $mito \\
    $peak \\
    $tss \\
    $excl_regs \\
    $autosom_ref \\
    --metrics-file "${prefix}.ataqv.json" \\
    --threads $task.cpus \\
    --name $prefix \\
    $organism \\
    $bam

cat <<-END_VERSIONS > versions.yml
"${task.process}":
    ataqv: \$( ataqv --version )
END_VERSIONS
"""

NextFlow ataqv From line 34 of ataqv/main.nf

"""
mkarv \\
    $args \\
    --concurrency $task.cpus \\
    --force \\
    ./html/ \\
    jsons/*

cat <<-END_VERSIONS > versions.yml
"${task.process}":
    # mkarv: \$( mkarv --version ) # Use this when version string has been fixed
    ataqv: \$( ataqv --version )
END_VERSIONS
"""

NextFlow ataqv From line 21 of mkarv/main.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 ${prefix}.${extension} -

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 44 of align/main.nf

"""
touch ${prefix}.${extension}
touch ${prefix}.bowtie2.log
touch ${prefix}.unmapped_1.fastq.gz
touch ${prefix}.unmapped_2.fastq.gz

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 From line 80 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 bowtie2
touch bowtie2/${fasta.baseName}.{1..4}.bt2
touch bowtie2/${fasta.baseName}.rev.{1,2}.bt2

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 32 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

"""
mkdir bwa

touch bwa/genome.amb
touch bwa/genome.ann
touch bwa/genome.bwt
touch bwa/genome.pac
touch bwa/genome.sa

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

NextFlow BWA From line 37 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}":
    getchromsizes: \$(echo \$(samtools --version 2>&1) | sed 's/^.*samtools //; s/Using.*\$//')
END_VERSIONS
"""

NextFlow SAMtools From line 24 of getchromsizes/main.nf

"""
touch ${fasta}.fai
touch ${fasta}.sizes

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

NextFlow From line 35 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

"""
printf "%s %s\\n" $rename_to | while read old_name new_name; do
    [ -f "\${new_name}" ] || ln -s \$old_name \$new_name
done

fastqc \\
    $args \\
    --threads $task.cpus \\
    $renamed_files

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

NextFlow FastQC From line 28 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 46 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

"""
# Not calling gunzip itself because it creates files
# with the original group ownership rather than the
# default one for that user / the work directory
gzip \\
    -cd \\
    $args \\
    $archive \\
    > $gunzip

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 41 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 28 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

"""
picard \\
    -Xmx${avail_mem}M \\
    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}M \\
    MarkDuplicates \\
    $args \\
    --INPUT $bam \\
    --OUTPUT ${prefix}.bam \\
    --REFERENCE_SEQUENCE $fasta \\
    --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 33 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 51 of markduplicates/main.nf

"""
picard \\
    -Xmx${avail_mem}M \\
    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} \\
    $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 \\
    --threads ${task.cpus-1} \\
    $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 25 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 41 of sort/main.nf

"""
samtools \\
    stats \\
    --threads ${task.cpus} \\
    ${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_list.join(' ')} \\
    --cores $cores \\
    --gzip \\
    ${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 42 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 \\
    ${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 57 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

"""
touch ${prefix}.bigWig

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

NextFlow From line 41 of bedgraphtobigwig/main.nf

"""
umi_tools \\
    extract \\
    -I $reads \\
    -S ${prefix}.umi_extract.fastq.gz \\
    $args \\
    > ${prefix}.umi_extract.log

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

NextFlow umi_tools From line 26 of extract/main.nf

"""
umi_tools \\
    extract \\
    -I ${reads[0]} \\
    --read2-in=${reads[1]} \\
    -S ${prefix}.umi_extract_1.fastq.gz \\
    --read2-out=${prefix}.umi_extract_2.fastq.gz \\
    $args \\
    > ${prefix}.umi_extract.log

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

NextFlow umi_tools From line 40 of extract/main.nf

"""
mkdir $prefix

## Ensures --strip-components only applied when top level of tar contents is a directory
## If just files or multiple directories, place all in prefix
if [[ \$(tar -taf ${archive} | grep -o -P "^.*?\\/" | uniq | wc -l) -eq 1 ]]; then
    tar \\
        -C $prefix --strip-components 1 \\
        -xavf \\
        $args \\
        $archive \\
        $args2
else
    tar \\
        -C $prefix \\
        -xavf \\
        $args \\
        $archive \\
        $args2
fi

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

"""
mkdir $prefix
touch ${prefix}/file.txt

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