A small-RNA sequencing analysis pipeline

public 1yr ago Version: 2.2.1 0 bookmarks

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Introduction

nf-core/smrnaseq is a bioinformatics best-practice analysis pipeline for Small RNA-Seq.

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 .

Online videos

A short talk about the history, current status and functionality on offer in this pipeline was given by Lorena Pantano (@lpantano) on 9th November 2021 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! )
1. Insert Size calculation
2. Collapse reads ( seqcluster )
Contamination filtering ( Bowtie2 )
Alignment against miRBase mature miRNA ( Bowtie1 )
Alignment against miRBase hairpin
1. Unaligned reads from step 3 ( Bowtie1 )
2. Collapsed reads from step 2.2 ( Bowtie1 )
Post-alignment processing of miRBase hairpin
1. Basic statistics from step 3 and step 4.1 ( SAMtools )
2. Analysis on miRBase, or MirGeneDB hairpin counts ( edgeR )
  - TMM normalization and a table of top expression hairpin
  - MDS plot clustering samples
  - Heatmap of sample similarities
3. miRNA and isomiR annotation from step 4.1 ( mirtop )
Alignment against host reference genome ( Bowtie1 )
1. Post-alignment processing of alignment against host reference genome ( SAMtools )
Novel miRNAs and known miRNAs discovery ( MiRDeep2 )
1. Mapping against reference genome with the mapper module
2. Known and novel miRNA discovery with the mirdeep2 module
miRNA quality control ( mirtrace )
Present QC for raw read, alignment, and expression results ( MultiQC )

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.

Now, you can run the pipeline using:

nextflow run nf-core/smrnaseq \
 -profile <docker/singularity/.../institute> \
 --input samplesheet.csv \
 --genome 'GRCh37' \
 --mirtrace_species 'hsa' \
 --protocol 'illumina' \
 --outdir <OUTDIR>

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, please refer to the usage documentation and the parameter documentation .

Pipeline output

To see the the results of a 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

nf-core/smrnaseq was originally written by P. Ewels, C. Wang, R. Hammarén, L. Pantano, A. Peltzer.

We thank the following people for their extensive assistance in the development of this pipeline:

Lorena Pantano ( @lpantano ) from MIT updated the pipeline to Nextflow DSL2.

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

Citations

If you use nf-core/smrnaseq for your analysis, please cite it using the following doi: 10.5281/zenodo.3456879

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

"""
echo $db_type
awk '/^>/ { x=index(\$6, "transcript_biotype:miRNA") } { if(!x) print }' $contaminants > subset.fa
blat -out=blast8 $mirna subset.fa /dev/stdout | awk 'BEGIN{FS="\t"}{if(\$11 < 1e-5)print \$1;}' | uniq > mirnahit.txt
awk 'BEGIN { while((getline<"mirnahit.txt")>0) l[">"\$1]=1 } /^>/ {x = l[\$1]} {if(!x) print }' subset.fa  > filtered.fa

cat <<-END_VERSIONS > versions.yml
"${task.process}":
    blat: \$(echo \$(blat) | grep Standalone | awk '{ if (match(\$0,/[0-9]*[0-9]/,m)) print m[0] }')
END_VERSIONS
"""

NextFlow From line 25 of local/blat_mirna.nf

"""
echo $db_type
awk '/^>/ { x=(index(\$6, "transcript_biotype:rRNA") || index(\$6, "transcript_biotype:miRNA")) } { if(!x) print }' $contaminants > subset.fa
blat -out=blast8 $mirna subset.fa /dev/stdout | awk 'BEGIN{FS="\t"}{if(\$11 < 1e-5)print \$1;}' | uniq > mirnahit.txt
awk 'BEGIN { while((getline<"mirnahit.txt")>0) l[">"\$1]=1 } /^>/ {x = l[\$1]} {if(!x) print }' subset.fa  > filtered.fa

cat <<-END_VERSIONS > versions.yml
"${task.process}":
    blat: \$(echo \$(blat) | grep Standalone | awk '{ if (match(\$0,/[0-9]*[0-9]/,m)) print m[0] }')
END_VERSIONS
"""

NextFlow From line 38 of local/blat_mirna.nf

        """
        echo $db_type
        blat -out=blast8 $mirna $contaminants /dev/stdout | awk 'BEGIN{FS="\t"}{if(\$11 < 1e-5)print \$1;}' | uniq > mirnahit.txt
        awk 'BEGIN { while((getline<"mirnahit.txt")>0) l[">"\$1]=1 } /^>/ {x = l[\$1]} {if(!x) print }' $contaminants  > filtered.fa

cat <<-END_VERSIONS > versions.yml
        "${task.process}":
            blat: \$(echo \$(blat) | grep Standalone | awk '{ if (match(\$0,/[0-9]*[0-9]/,m)) print m[0] }')
        END_VERSIONS
        """

NextFlow From line 51 of local/blat_mirna.nf

"""
bowtie2-build ${fasta} fasta_bidx --threads ${task.cpus}

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 20 of local/bowtie_contaminants.nf

"""
# Remove any special base characters from reference genome FASTA file
sed '/^[^>]/s/[^ATGCatgc]/N/g' $fasta > genome.edited.fa
sed -i 's/ .*//' genome.edited.fa

# Build bowtie index
bowtie-build genome.edited.fa genome --threads ${task.cpus}

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

NextFlow Bowtie 2 From line 22 of local/bowtie_genome.nf

"""
INDEX=`find -L ./ -name "*.3.ebwt" | sed 's/.3.ebwt//'`
bowtie2 \\
    --threads ${task.cpus} \\
    --very-sensitive-local \\
    -k 1 \\
    -x \$INDEX \\
    --un ${meta.id}.${contaminant_type}.filter.unmapped.contaminant.fastq \\
    ${reads} \\
    ${args} \\
    -S ${meta.id}.filter.contaminant.sam > ${meta.id}.contaminant_bowtie.log 2>&1

# extracting number of reads from bowtie logs
awk -v type=${contaminant_type} 'BEGIN{tot=0} {if(NR==4 || NR == 5){tot += \$1}} END {print "\\""type"\\": "tot }' ${meta.id}.contaminant_bowtie.log | tr -d , > filtered.${meta.id}_${contaminant_type}.stats

cat <<-END_VERSIONS > versions.yml
"${task.process}":
    bowtie2: \$(echo \$(bowtie2 --version 2>&1) | sed 's/^.*bowtie2-align-s version //; s/ .*\$//' | tr -d '\0')
END_VERSIONS
"""

NextFlow Bowtie 2 From line 24 of local/bowtie_map_contaminants.nf

"""
INDEX=`find -L ./ -name "*.3.ebwt" | sed 's/.3.ebwt//'`
bowtie \\
    -x \$INDEX \\
    -q <(zcat $reads) \\
    -p ${task.cpus} \\
    -t \\
    -k 50 \\
    --best \\
    --strata \\
    -e 99999 \\
    --chunkmbs 2048 \\
    --un ${meta.id}_unmapped.fq -S > ${meta.id}.sam

samtools view -bS ${meta.id}.sam > ${meta.id}.bam

if [ ! -f  "${meta.id}_unmapped.fq" ]
then
    touch ${meta.id}_unmapped.fq
fi
gzip ${meta.id}_unmapped.fq
mkdir unmapped
mv  ${meta.id}_unmapped.fq.gz  unmapped/.

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

NextFlow SAMtools Bowtie 2 From line 23 of local/bowtie_map_mirna.nf

"""
bowtie-build ${fasta} fasta_bidx --threads ${task.cpus}

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

NextFlow Bowtie 2 From line 20 of local/bowtie_mirna.nf

"""
collapse_mirtop.r ${mirtop}

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/datatable_merge.nf

"""
edgeR_miRBase.r $input_files

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

NextFlow limma From line 20 of local/edger_qc.nf

"""
readnumber=\$(wc -l ${reads} | awk '{ print \$1/4 }')
cat ./filtered.${meta.id}_*.stats | \\
tr '\n' ', ' | \\
awk -v sample=${meta.id} -v readnumber=\$readnumber '{ print "id: \\"my_pca_section\\"\\nsection_name: \\"Contamination Filtering\\"\\ndescription: \\"This plot shows the amount of reads filtered by contaminant type.\\"\\nplot_type: \\"bargraph\\"\\npconfig:\\n  id: \\"contamination_filter_plot\\"\\n  title: \\"Contamination Plot\\"\\n  ylab: \\"Number of reads\\"\\ndata:\\n    "sample": {"\$0"\\"remaining reads\\": "readnumber"}" }' > ${meta.id}.contamination_mqc.yaml
gzip -c ${reads} > ${meta.id}.filtered.fastq.gz
"""

NextFlow From line 21 of local/filter_stats.nf

"""
fasta_formatter -w 0 -i $fasta -o ${fasta}_idx.fa

cat <<-END_VERSIONS > versions.yml
"${task.process}":
    fastx_toolkit:  \$(echo "$VERSION")
END_VERSIONS
"""

NextFlow From line 23 of local/format_fasta_mirna.nf

"""
mapper.pl \\
$reads \\
    -e \\
    -h \\
    -i \\
    -j \\
    -m \\
    -p $index_base \\
    -s ${meta.id}_collapsed.fa \\
    -t ${meta.id}_reads_vs_refdb.arf \\
    -o 4

cat <<-END_VERSIONS > versions.yml
"${task.process}":
    mapper: \$(echo "$VERSION")
END_VERSIONS
"""

NextFlow mirdeep2 From line 25 of local/mirdeep2_mapper.nf

"""
pigz -f -d -p $task.cpus $reads

cat <<-END_VERSIONS > versions.yml
${task.process}":
    pigz: \$( pigz --version 2>&1 | sed 's/pigz //g' )
END_VERSIONS
"""

NextFlow From line 22 of local/mirdeep2_prepare.nf

"""
miRDeep2.pl  \\
    $reads   \\
    $fasta   \\
    $arf     \\
    $mature  \\
    none     \\
    $hairpin \\
    -d       \\
    -z _${reads.simpleName}

cat <<-END_VERSIONS > versions.yml
${task.process}":
    mirdeep2: \$(echo "$VERSION")
END_VERSIONS
"""

NextFlow From line 26 of local/mirdeep2_run.nf

"""
mirtop gff --hairpin $hairpin --gtf $gtf -o mirtop --sps $filter_species ./bams/*
mirtop counts --hairpin $hairpin --gtf $gtf -o mirtop --sps $filter_species --add-extra --gff mirtop/mirtop.gff
mirtop export --format isomir --hairpin $hairpin --gtf $gtf --sps $filter_species -o mirtop mirtop/mirtop.gff
mirtop stats mirtop/mirtop.gff --out mirtop/stats
mv mirtop/stats/mirtop_stats.log mirtop/stats/full_mirtop_stats.log

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

NextFlow mirtop From line 26 of local/mirtop_quant.nf

"""
export mirtracejar=\$(dirname \$(which mirtrace))

${config_lines.join("\n    ")}

java $java_mem -jar \$mirtracejar/mirtrace.jar --mirtrace-wrapper-name mirtrace qc  \\
    --species $params.mirtrace_species \\
    $primer \\
    $protocol \\
    --config mirtrace_config \\
    --write-fasta \\
    --output-dir mirtrace \\
    --force

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

NextFlow mirtrace From line 31 of local/mirtrace.nf

"""
# Uncompress FASTA reference files if necessary
FASTA="$fasta"
if [ \${FASTA: -3} == ".gz" ]; then
    gunzip -f \$FASTA
    FASTA=\${FASTA%%.gz}
fi
sed 's/&gt;/>/g' \$FASTA | sed 's#<br>#\\n#g' | sed 's#</p>##g' | sed 's#<p>##g' > \${FASTA}_html_cleaned.fa
# Remove spaces from miRBase FASTA files
sed '#^[^>]#s#[^AUGCaugc]#N#g' \${FASTA}_html_cleaned.fa > \${FASTA}_parsed.fa

sed -i 's#\s.*##' \${FASTA}_parsed.fa
seqkit grep -r --pattern \".*${filter_species}-.*\" \${FASTA}_parsed.fa > \${FASTA}_sps.fa
seqkit seq --rna2dna \${FASTA}_sps.fa > \${FASTA}_igenome.fa

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

NextFlow seqkit From line 21 of local/parse_fasta_mirna.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 21 of local/samplesheet_check.nf

"""
seqcluster collapse -f $reads -m 1 --min_size 15 -o collapsed
gzip collapsed/*_trimmed.fastq
mkdir final
mv collapsed/*.fastq.gz final/.

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

NextFlow seqcluster From line 21 of local/seqcluster_collapse.nf

"""
cat ${readList.join(' ')} > ${prefix}.merged.fastq.gz

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

NextFlow From line 26 of fastq/main.nf

"""
cat ${read1.join(' ')} > ${prefix}_1.merged.fastq.gz
cat ${read2.join(' ')} > ${prefix}_2.merged.fastq.gz

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

NextFlow From line 40 of fastq/main.nf

"""
touch ${prefix}.merged.fastq.gz

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

NextFlow From line 57 of fastq/main.nf

"""
touch ${prefix}_1.merged.fastq.gz
touch ${prefix}_2.merged.fastq.gz

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

NextFlow From line 68 of fastq/main.nf

"""
[ ! -f  ${prefix}.fastq.gz ] && ln -sf $reads ${prefix}.fastq.gz

fastp \\
    --stdout \\
    --in1 ${prefix}.fastq.gz \\
    --thread $task.cpus \\
    --json ${prefix}.fastp.json \\
    --html ${prefix}.fastp.html \\
    $adapter_list \\
    $fail_fastq \\
    $args \\
    2> ${prefix}.fastp.log \\
| gzip -c > ${prefix}.fastp.fastq.gz

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

NextFlow fastp From line 36 of fastp/main.nf

"""
[ ! -f  ${prefix}.fastq.gz ] && ln -sf $reads ${prefix}.fastq.gz

fastp \\
    --in1 ${prefix}.fastq.gz \\
    --out1  ${prefix}.fastp.fastq.gz \\
    --thread $task.cpus \\
    --json ${prefix}.fastp.json \\
    --html ${prefix}.fastp.html \\
    $adapter_list \\
    $fail_fastq \\
    $args \\
    2> ${prefix}.fastp.log

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

NextFlow fastp From line 57 of fastp/main.nf

"""
[ ! -f  ${prefix}_1.fastq.gz ] && ln -sf ${reads[0]} ${prefix}_1.fastq.gz
[ ! -f  ${prefix}_2.fastq.gz ] && ln -sf ${reads[1]} ${prefix}_2.fastq.gz
fastp \\
    --in1 ${prefix}_1.fastq.gz \\
    --in2 ${prefix}_2.fastq.gz \\
    --out1 ${prefix}_1.fastp.fastq.gz \\
    --out2 ${prefix}_2.fastp.fastq.gz \\
    --json ${prefix}.fastp.json \\
    --html ${prefix}.fastp.html \\
    $adapter_list \\
    $fail_fastq \\
    $merge_fastq \\
    --thread $task.cpus \\
    --detect_adapter_for_pe \\
    $args \\
    2> ${prefix}.fastp.log

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

NextFlow fastp From line 78 of fastp/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

"""
multiqc \\
    --force \\
    $args \\
    $config \\
    $extra_config \\
    .

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

NextFlow MultiQC From line 28 of multiqc/main.nf

"""
touch multiqc_data
touch multiqc_plots
touch multiqc_report.html

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

NextFlow MultiQC From line 43 of multiqc/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

"""
touch ${prefix}.flagstat

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 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

"""
touch ${prefix}.idxstats

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

NextFlow From line 40 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
"""