circRNA quantification, differential expression analysis and miRNA target prediction of RNA-Seq data

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Introduction

nf-core/circrna is a bioinformatics best-practice analysis pipeline for circRNA quantification, differential expression analysis and miRNA target prediction of RNA-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

Raw read QC ( FastQC )
Adapter trimming ( Trim Galore! )
circRNA quantification
circRNA annotation
Export mature spliced length as FASTA file
Annotate parent gene, underlying transcripts.
circRNA count matrix
miRNA target prediction
1. miRanda
2. TargetScan
3. Filter results, miRNAs must be called by both tools
Differential expression analysis DESeq2
Circular - Linear ratio tests 'CircTest'
MultiQC report MultiQC

Quick Start

Install Nextflow ( >=22.10.1 )
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/circrna -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/circrna --input samplesheet.csv --outdir <OUTDIR> --genome GRCh37 -profile <docker/singularity/podman/shifter/charliecloud/conda/institute> --tool 'ciriquant' --module 'circrna_discovery,mirna_prediction,differential_expression' --bsj_reads 2

Documentation

The nf-core/circrna pipeline comes with documentation about the pipeline usage , parameters and output .

Credits

nf-core/circrna was originally written by Barry Digby.

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

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

Citations

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

"""
grep -vf ${workflow.projectDir}/bin/unwanted_biotypes.txt $gtf > filt.gtf
mv $bed circs.bed

annotate_outputs.sh $exon_boundary &> ${prefix}.log
mv master_bed12.bed ${prefix}.bed.tmp

awk -v FS="\t" '{print \$11}' ${prefix}.bed.tmp > mature_len.tmp
awk -v FS="," '{for(i=t=0;i<NF;) t+=\$++i; \$0=t}1' mature_len.tmp > mature_length

paste ${prefix}.bed.tmp mature_length > ${prefix}.bed

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

NextFlow BEDTools From line 27 of full_annotation/main.nf

"""
# remove redundant biotypes from GTF.
grep -vf ${workflow.projectDir}/bin/unwanted_biotypes.txt $gtf > filt.gtf

# generate circrna BED file.
tail -n +2 $circrna_matrix | awk '{print \$1}' > IDs.txt
ID_to_BED.sh IDs.txt
cat *.bed > merged.txt && rm IDs.txt && rm *.bed && mv merged.txt circs.bed

# Re-use annotation script to identify the host gene.
annotate_outputs.sh $exon_boundary &> annotation.log
awk -v OFS="\t" '{print \$4, \$14}' master_bed12.bed > circrna_host-gene.txt

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

NextFlow BEDTools From line 23 of parent_gene/main.nf

"""
awk '{if(\$13 >= ${bsj_reads}) print \$0}' ${prefix}.txt | awk -v OFS="\t" '{print \$1,\$2,\$3,\$6,\$13}' > ${prefix}_${meta.tool}.bed

awk -v OFS="\t" '{print \$1, \$2, \$3, \$1":"\$2"-"\$3":"\$4, \$5, \$4}' ${prefix}_${meta.tool}.bed > ${prefix}_${meta.tool}_circs.bed
"""

NextFlow From line 19 of filter/main.nf

"""
gtfToGenePred \
    $args \
    $gtf \
    ${prefix}.genepred

awk -v OFS="\t" '{print \$12, \$1, \$2, \$3, \$4, \$5, \$6, \$7, \$8, \$9, \$10}' ${prefix}.genepred > ${prefix}.txt

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

NextFlow gtftogenepred From line 24 of reference/main.nf

"""
mkdir -p star_dir && mv *.tab *.junction *.sam star_dir
postProcessStarAlignment.pl --starDir star_dir/ --outDir ./

awk '{if(\$5 >= ${bsj_reads}) print \$0}' ${prefix}.filteredJunctions.bed | awk  -v OFS="\t" -F"\t" '{print \$1,\$2,\$3,\$6,\$5}' > ${prefix}_circrna_finder.bed

awk -v OFS="\t" '{print \$1, \$2, \$3, \$1":"\$2"-"\$3":"\$4, \$5, \$4}' ${prefix}_circrna_finder.bed > ${prefix}_circrna_finder_circs.bed

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

NextFlow From line 27 of filter/main.nf

"""
prepare_circ_test.R

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 prepare/main.nf

"""
circ_test.R $circ_csv $linear_csv $phenotype

cat <<-END_VERSIONS > versions.yml
"${task.process}":
    r-base: \$(echo \$(R --version 2>&1) | sed 's/^.*R version //; s/ .*\$//')
    aod: \$(Rscript -e "library(aod); cat(as.character(packageVersion('aod')))")
    ggplot2: \$(Rscript -e "library(ggplot2); cat(as.character(packageVersion('ggplot2')))")
    plyr: \$(Rscript -e "library(plyr); cat(as.character(packageVersion('plyr')))")
END_VERSIONS
"""

NextFlow ggplot2 From line 22 of test/main.nf

"""
CIRIquant \\
    -t ${task.cpus} \\
    -1 ${reads[0]} \\
    -2 ${reads[1]} \\
    --config $yml \\
    --no-gene \\
    -o ${prefix} \\
    -p ${prefix}

cat <<-END_VERSIONS > versions.yml
"${task.process}":
    bwa: \$(echo \$(bwa 2>&1) | sed 's/^.*Version: //; s/Contact:.*\$//')
    ciriquant : \$(echo \$(CIRIquant --version 2>&1) | sed 's/CIRIquant //g' )
    samtools: \$(echo \$(samtools --version 2>&1) | sed 's/^.*samtools //; s/Using.*\$//')
    stringtie: \$(stringtie --version 2>&1)
    hisat2: $VERSION
END_VERSIONS
"""

NextFlow ciriquant From line 26 of ciriquant/main.nf

"""
grep -v "#" ${prefix}.gtf | awk '{print \$14}' | cut -d '.' -f1 > counts
grep -v "#" ${prefix}.gtf | awk -v OFS="\t" '{print \$1,\$4,\$5,\$7}' > ${prefix}.tmp
paste ${prefix}.tmp counts > ${prefix}_unfilt.bed

awk '{if(\$5 >= ${bsj_reads}) print \$0}' ${prefix}_unfilt.bed > ${prefix}_filt.bed
grep -v '^\$' ${prefix}_filt.bed > ${prefix}_ciriquant

awk -v OFS="\t" '{\$2-=1;print}' ${prefix}_ciriquant > ${prefix}_ciriquant.bed
rm ${prefix}.gtf

awk -v OFS="\t" '{print \$1, \$2, \$3, \$1":"\$2"-"\$3":"\$4, \$5, \$4}' ${prefix}_ciriquant.bed > ${prefix}_ciriquant_circs.bed
"""

NextFlow From line 18 of filter/main.nf

"""
BWA=`which bwa`
HISAT2=`which hisat2`
STRINGTIE=`which stringtie`
SAMTOOLS=`which samtools`

touch travis.yml
printf "name: ciriquant\ntools:\n  bwa: \$BWA\n  hisat2: \$HISAT2\n  stringtie: \$STRINGTIE\n  samtools: \$SAMTOOLS\n\nreference:\n  fasta: ${fasta_path}\n  gtf: ${gtf_path}\n  bwa_index: ${bwa_path}/${bwa_prefix}\n  hisat_index: ${hisat2_path}/${hisat2_prefix}" >> travis.yml
"""

NextFlow From line 28 of yml/main.nf

"""
python ${workflow.projectDir}/bin/circRNA_counts_matrix.py > matrix.txt
## handle non-canon chromosomes here (https://stackoverflow.com/questions/71479919/joining-columns-based-on-number-of-fields)
n_samps=\$(ls *.bed | wc -l)
canon=\$(awk -v a="\$n_samps" 'BEGIN {print a + 4}')
awk -v n="\$canon" '{ for (i = 2; i <= NF - n + 1; ++i) { \$1 = \$1"-"\$i; \$i=""; } } 1' matrix.txt | awk -v OFS="\t" '\$1=\$1' > circRNA_matrix.txt
Rscript ${workflow.projectDir}/bin/reformat_count_matrix.R

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

NextFlow From line 21 of combined/main.nf

"""
## make list of files for R to read
ls *.bed > samples.csv

## Add catch for empty bed file and delete
bash ${workflow.projectDir}/bin/check_empty.sh

## Use intersection of "n" (params.tool_filter) circRNAs called by tools
## remove duplicate IDs, keep highest count.
Rscript ${workflow.projectDir}/bin/consolidate_algorithms_intersection.R samples.csv $tool_filter $duplicates_fun
mv combined_counts.bed ${prefix}.bed

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

NextFlow From line 24 of merge_tools/main.nf

"""
# Strip tool name from BED files (no consolidation prior to this step for 1 tool)
for b in *.bed; do
    basename=\${b%".bed"};
    sample_name=\${basename%"_${tool_name}"};
    mv \$b \${sample_name}.bed
done

python ${workflow.projectDir}/bin/circRNA_counts_matrix.py > matrix.txt
## handle non-canon chromosomes here (https://stackoverflow.com/questions/71479919/joining-columns-based-on-number-of-fields)
n_samps=\$(ls *.bed | wc -l)
canon=\$(awk -v a="\$n_samps" 'BEGIN {print a + 4}')
awk -v n="\$canon" '{ for (i = 2; i <= NF - n + 1; ++i) { \$1 = \$1"-"\$i; \$i=""; } } 1' matrix.txt | awk -v OFS="\t" '\$1=\$1' > circRNA_matrix.txt
Rscript ${workflow.projectDir}/bin/reformat_count_matrix.R

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

NextFlow From line 23 of single/main.nf

"""
sed -i 's/^chr//g' $gtf

mkdir ${prefix} && mv ${prefix}.Chimeric.out.junction ${prefix} && printf "${prefix}/${prefix}.Chimeric.out.junction" > samplesheet

DCC @samplesheet -D -an $gtf -Pi -ss -F -M -Nr 1 1 -fg -A $fasta -N -T ${task.cpus}

awk '{print \$6}' CircCoordinates >> strand
paste CircRNACount strand | tail -n +2 | awk -v OFS="\t" '{print \$1,\$2,\$3,\$5,\$4}' >> ${prefix}.txt

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

NextFlow dcc From line 26 of dcc/main.nf

"""
sed -i 's/^chr//g' $gtf

mkdir ${prefix} && mv ${prefix}.Chimeric.out.junction ${prefix} && printf "${prefix}/${prefix}.Chimeric.out.junction" > samplesheet
mkdir ${prefix}_mate1 && mv ${prefix}_mate1.Chimeric.out.junction ${prefix}_mate1 && printf "${prefix}_mate1/${prefix}_mate1.Chimeric.out.junction" > mate1file
mkdir ${prefix}_mate2 && mv ${prefix}_mate2.Chimeric.out.junction ${prefix}_mate2 && printf "${prefix}_mate2/${prefix}_mate2.Chimeric.out.junction" > mate2file

DCC @samplesheet -mt1 @mate1file -mt2 @mate2file -D -an $gtf -Pi -ss -F -M -Nr 1 1 -fg -A $fasta -N -T ${task.cpus}

awk '{print \$6}' CircCoordinates >> strand
paste CircRNACount strand | tail -n +2 | awk -v OFS="\t" '{print \$1,\$2,\$3,\$5,\$4}' >> ${prefix}.txt

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

NextFlow dcc From line 42 of dcc/main.nf

"""
awk '{if(\$5 >= ${bsj_reads}) print \$0}' ${prefix}.txt > ${prefix}_dcc.filtered
awk -v OFS="\t" '{\$2-=1;print}' ${prefix}_dcc.filtered > ${prefix}_dcc.bed
awk -v OFS="\t" '{print \$1, \$2, \$3, \$1":"\$2"-"\$3":"\$4, \$5, \$4}' ${prefix}_dcc.bed > ${prefix}_dcc_circs.bed
"""

NextFlow From line 18 of filter/main.nf

"""
## prepDE && circRNA counts headers are sorted such that uppercase preceedes lowercase i.e Z before a
## reformat the phenotype file to match the order of the samples.
head -n 1 $phenotype > header
tail -n +2 $phenotype | LC_COLLATE=C sort > sorted_pheno
cat header sorted_pheno > tmp && rm phenotype.csv && mv tmp phenotype.csv

DEA.R $gene_matrix $phenotype $circrna_matrix $species ensembl_database_map.txt
mv boxplots/ circRNA/

cat <<-END_VERSIONS > versions.yml
"${task.process}":
    r-base: \$(echo \$(R --version 2>&1) | sed 's/^.*R version //; s/ .*\$//')
    argparser: \$(Rscript -e "library(argparser); cat(as.character(packageVersion('argparser')))")
    biomart: \$(Rscript -e "library(biomaRt); cat(as.character(packageVersion('biomaRt')))")
    deseq2: \$(Rscript -e "library(DESeq2); cat(as.character(packageVersion('DESeq2')))")
    dplyr: \$(Rscript -e "library(dplyr); cat(as.character(packageVersion('dplyr')))")
    enhancedvolcano: \$(Rscript -e "library(EnhancedVolcano); cat(as.character(packageVersion('EnhancedVolcano')))")
    gplots: \$(Rscript -e "library(gplots); cat(as.character(packageVersion('gplots')))")
    ggplot2: \$(Rscript -e "library(ggplot2); cat(as.character(packageVersion('ggplot2')))")
    ggpubr: \$(Rscript -e "library(ggpubr); cat(as.character(packageVersion('ggpubr')))")
    ihw: \$(Rscript -e "library(IHW); cat(as.character(packageVersion('IHW')))")
    pvclust: \$(Rscript -e "library(pvclust); cat(as.character(packageVersion('pvclust')))")
    pcatools: \$(Rscript -e "library(PCAtools); cat(as.character(packageVersion('PCAtools')))")
    pheatmap: \$(Rscript -e "library(pheatmap); cat(as.character(packageVersion('pheatmap')))")
    rcolorbrewer: \$(Rscript -e "library(RColorBrewer); cat(as.character(packageVersion('RColorBrewer')))")
END_VERSIONS
"""

NextFlow ggplot2 DESeq2 pheatmap From line 27 of differential_expression/main.nf

"""
## FASTA sequences (bedtools does not like the extra annotation info - split will not work properly)
cut -d\$'\t' -f1-12 ${prefix}.bed > bed12.tmp
bedtools getfasta -fi $fasta -bed bed12.tmp -s -split -name > circ_seq.tmp

## clean fasta header
grep -A 1 '>' circ_seq.tmp | cut -d: -f1,2,3 > ${prefix}.fa && rm circ_seq.tmp

## add backsplice sequence for miRanda Targetscan, publish canonical FASTA to results.
rm $fasta
bash ${workflow.projectDir}/bin/backsplice_gen.sh ${prefix}.fa

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

NextFlow BEDTools From line 26 of fasta/main.nf

"""
unmapped2anchors.py $bam | gzip > ${prefix}_anchors.qfa.gz

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

NextFlow From line 23 of anchors/main.nf

"""
grep CIRCULAR $bed | \
    grep -v chrM | \
    awk '\$5>=${bsj_reads}' | \
    grep UNAMBIGUOUS_BP | grep ANCHOR_UNIQUE | \
    maxlength.py 100000 \
    > ${prefix}.txt

tail -n +2 ${prefix}.txt | awk -v OFS="\t" '{print \$1,\$2,\$3,\$6,\$5}' > ${prefix}_find_circ.bed

awk -v OFS="\t" '{print \$1, \$2, \$3, \$1":"\$2"-"\$3":"\$4, \$5, \$4}' ${prefix}_find_circ.bed > ${prefix}_find_circ_circs.bed

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

NextFlow From line 25 of filter/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 \\
    --threads $task.cpus \\
    --reorder \\
    --mm \\
    -D 20 \\
    --score-min=C,-15,0 \\
    -q \\
    -x \$INDEX \\
    -U $anchors | \\
    find_circ.py  --genome=$fasta --prefix=${prefix} --stats=${prefix}.sites.log --reads=${prefix}.sites.reads > ${prefix}.sites.bed

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

NextFlow Bowtie 2 From line 27 of find_circ/main.nf

"""
$handleGzip_R1

mapsplice.py \\
    -c $chromosomes \\
    -x $gtf_prefix \\
    -1 ${read1} \\
    -p ${task.cpus} \\
    --bam \\
    --gene-gtf $gtf \\
    -o $prefix \\
    $args

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

NextFlow MapSplice From line 31 of align/main.nf

"""
$handleGzip_R1
$handleGzip_R2

mapsplice.py \\
    -c $chromosomes \\
    -x $gtf_prefix \\
    -1 ${read1} \\
    -2 ${read2} \\
    -p ${task.cpus} \\
    --bam \\
    --gene-gtf $gtf \\
    -o $prefix \\
    $args

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

NextFlow MapSplice From line 54 of align/main.nf

"""
## reformat and sort miRanda, TargetScan outputs, convert to BED for overlaps.
tail -n +2 $targetscan | sort -k1,1 -k4n | awk -v OFS="\t" '{print \$1, \$2, \$4, \$5, \$9}' | awk -v OFS="\t" '{print \$2, \$3, \$4, \$1, "0", \$5}' > targetscan.bed
tail -n +2 $miranda | sort -k2,2 -k7n | awk -v OFS="\t" '{print \$2, \$1, \$3, \$4, \$7, \$8}' | awk -v OFS="\t" '{print \$2, \$5, \$6, \$1, \$3, \$4}' | sed 's/^[^-]*-//g' > miranda.bed

## intersect, consolidate miRanda, TargetScan information about miRs.
## -wa to output miRanda hits - targetscan makes it difficult to resolve duplicate miRNAs at MRE sites.
bedtools intersect -a miranda.bed -b targetscan.bed -wa > ${prefix}.mirnas.tmp
bedtools intersect -a targetscan.bed -b miranda.bed | awk '{print \$6}' > mirna_type

## remove duplicate miRNA entries at MRE sites.
## strategy: sory by circs, sort by start position, sort by site type - the goal is to take the best site type (i.e rank site type found at MRE site).
paste ${prefix}.mirnas.tmp mirna_type | sort -k3,3 -k2n -k7r | awk -v OFS="\t" '{print \$4,\$1,\$2,\$3,\$5,\$6,\$7}' | awk -F "\t" '{if (!seen[\$1,\$2,\$3,\$4,\$5,\$6]++)print}' | sort -k1,1 -k3n > ${prefix}.mirna_targets.tmp
echo -e "circRNA\tmiRNA\tStart\tEnd\tScore\tEnergy_KcalMol\tSite_type" | cat - ${prefix}.mirna_targets.tmp > ${prefix}.mirna_targets.txt

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

NextFlow BEDTools From line 23 of mirna_targets/main.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

"""
grep ';C;' ${prefix}.sngl.bed | awk -v OFS="\t" '{print \$1,\$2,\$3,\$6}' | sort | uniq -c | awk -v OFS="\t" '{print \$2,\$3,\$4,\$5,\$1}' > ${prefix}_collapsed.bed

awk -v OFS="\t" -v BSJ=${bsj_reads} '{if(\$5>=BSJ) print \$0}' ${prefix}_collapsed.bed > ${prefix}_segemehl.bed

awk -v OFS="\t" '{print \$1, \$2, \$3, \$1":"\$2"-"\$3":"\$4, \$5, \$4}' ${prefix}_segemehl.bed > ${prefix}_segemehl_circs.bed
"""

NextFlow From line 19 of filter/main.nf

"""
cat *.tab | awk -v BSJ=${bsj_reads} '(\$7 >= BSJ && \$6==0)' | cut -f1-6 | sort | uniq > dataset.SJ.out.tab
"""

NextFlow From line 15 of sjdb/main.nf

"""
for file in \$(ls *.gtf); do sample_id=\${file%".transcripts.gtf"}; touch samples.txt; printf "\$sample_id\t\$file\n" >> samples.txt ; done

prepDE.py -i samples.txt
"""

NextFlow From line 20 of prepde/main.nf

"""
bash ${workflow.projectDir}/bin/targetscan_format.sh $mature
"""

NextFlow From line 15 of database/main.nf

"""
##format for targetscan
cat $fasta | grep ">" | sed 's/>//g' > id
cat $fasta | grep -v ">" > seq
paste id seq | awk -v OFS="\t" '{print \$1, "0000", \$2}' > ${prefix}_ts.txt
# run targetscan
targetscan_70.pl mature.txt ${prefix}_ts.txt ${prefix}.txt

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

NextFlow From line 24 of predict/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}.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 bowtie
bowtie-build --threads $task.cpus $fasta bowtie/${fasta.baseName}
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 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

"""
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

"""
CIRCexplorer2 \\
    annotate \\
    -r $gene_annotation \\
    -g $fasta \\
    -b $junctions \\
    -o ${prefix}.txt \\
    $args

cat <<-END_VERSIONS > versions.yml
"${task.process}":
    circexplorer2: \$(echo \$(CIRCexplorer2 --version 2>&1) )
END_VERSIONS
"""

NextFlow circexplorer2 From line 25 of annotate/main.nf

"""
touch ${prefix}.txt

cat <<-END_VERSIONS > versions.yml
"${task.process}":
    circexplorer2: \$(echo \$(CIRCexplorer2 --version 2>&1) )
END_VERSIONS
"""

NextFlow From line 42 of annotate/main.nf

"""
CIRCexplorer2 \\
    parse \\
    $aligner \\
    $fusions \\
    -b ${prefix}.bed \\
    $args

cat <<-END_VERSIONS > versions.yml
"${task.process}":
    circexplorer2: \$( echo \$(CIRCexplorer2 --version 2>&1) )
END_VERSIONS
"""

NextFlow circexplorer2 From line 25 of parse/main.nf

"""
touch ${prefix}.bed

cat <<-END_VERSIONS > versions.yml
"${task.process}":
    circexplorer2: \$( echo \$(CIRCexplorer2 --version 2>&1) )
END_VERSIONS
"""

NextFlow From line 41 of parse/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 42 of fastqc/main.nf

"""
INDEX=`find -L ./ -name "*.1.ht2" | sed 's/\\.1.ht2\$//'`
hisat2 \\
    -x \$INDEX \\
    -U $reads \\
    $strandedness \\
    --known-splicesite-infile $splicesites \\
    --summary-file ${prefix}.hisat2.summary.log \\
    --threads $task.cpus \\
    $seq_center \\
    $unaligned \\
    $args \\
    | samtools view -bS -F 4 -F 256 - > ${prefix}.bam

cat <<-END_VERSIONS > versions.yml
"${task.process}":
    hisat2: $VERSION
    samtools: \$(echo \$(samtools --version 2>&1) | sed 's/^.*samtools //; s/Using.*\$//')
END_VERSIONS
"""

NextFlow SAMtools HISAT2 From line 39 of align/main.nf

"""
INDEX=`find -L ./ -name "*.1.ht2" | sed 's/\\.1.ht2\$//'`
hisat2 \\
    -x \$INDEX \\
    -1 ${reads[0]} \\
    -2 ${reads[1]} \\
    $strandedness \\
    --known-splicesite-infile $splicesites \\
    --summary-file ${prefix}.hisat2.summary.log \\
    --threads $task.cpus \\
    $seq_center \\
    $unaligned \\
    --no-mixed \\
    --no-discordant \\
    $args \\
    | samtools view -bS -F 4 -F 8 -F 256 - > ${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}":
    hisat2: $VERSION
    samtools: \$(echo \$(samtools --version 2>&1) | sed 's/^.*samtools //; s/Using.*\$//')
END_VERSIONS
"""

NextFlow SAMtools HISAT2 From line 61 of align/main.nf

"""
mkdir hisat2
$extract_exons
hisat2-build \\
    -p $task.cpus \\
    $ss \\
    $exon \\
    $args \\
    $fasta \\
    hisat2/${fasta.baseName}

cat <<-END_VERSIONS > versions.yml
"${task.process}":
    hisat2: $VERSION
END_VERSIONS
"""

NextFlow HISAT2 From line 48 of build/main.nf

"""
hisat2_extract_splice_sites.py $gtf > ${gtf.baseName}.splice_sites.txt
cat <<-END_VERSIONS > versions.yml
"${task.process}":
    hisat2: $VERSION
END_VERSIONS
"""

NextFlow From line 24 of extractsplicesites/main.nf

"""
miranda \\
    $mirbase \\
    $query \\
    $args \\
    -out ${prefix}.out

echo "miRNA\tTarget\tScore\tEnergy_KcalMol\tQuery_Start\tQuery_End\tSubject_Start\tSubject_End\tAln_len\tSubject_Identity\tQuery_Identity" > ${prefix}.txt
grep -A 1 "Scores for this hit:" ${prefix}.out | sort | grep ">"  | cut -c 2- | tr ' ' '\t' >> ${prefix}.txt

cat <<-END_VERSIONS > versions.yml
"${task.process}":
    miranda: \$(echo \$(miranda -v | sed -n 4p | sed 's/^.*miranda v//; s/microRNA.*\$//' ))
END_VERSIONS
"""

NextFlow miranda From line 24 of miranda/main.nf

"""
touch ${prefix}.txt

cat <<-END_VERSIONS > versions.yml
"${task.process}":
    miranda: \$(echo \$(miranda -v | sed -n 4p | sed 's/^.*miranda v//; s/microRNA.*\$//' ))
END_VERSIONS
"""

NextFlow From line 42 of miranda/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 \\
    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 35 of sort/main.nf

"""
samtools \\
    view \\
    --threads ${task.cpus-1} \\
    ${reference} \\
    ${readnames} \\
    $args \\
    -o ${prefix}.${file_type} \\
    $input \\
    $args2

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

NextFlow SAMtools From line 38 of view/main.nf

"""
touch ${prefix}.bam
touch ${prefix}.cram

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

NextFlow From line 57 of view/main.nf

"""
mkdir -p $prefix

segemehl.x \\
    -t $task.cpus \\
    -d $fasta \\
    -i $index \\
    $reads \\
    $args \\
    -o ${prefix}/${prefix}.${suffix}

cat <<-END_VERSIONS > versions.yml
"${task.process}":
    segemehl: \$(echo \$(segemehl.x 2>&1 | grep "ge5dee" | awk -F Z '{print substr(\$1, 2, 6)}' ))
END_VERSIONS
"""

NextFlow From line 27 of align/main.nf

"""
mkdir -p $prefix
touch ${prefix}/${prefix}.${suffix}

cat <<-END_VERSIONS > versions.yml
"${task.process}":
    segemehl: \$(echo \$(segemehl.x 2>&1 | grep "ge5dee" | awk -F Z '{print substr(\$1, 2, 6)}' ))
END_VERSIONS
"""

NextFlow From line 47 of align/main.nf

"""
segemehl.x \\
    -t $task.cpus \\
    -d $fasta \\
    -x ${prefix}.idx \\
    $args

cat <<-END_VERSIONS > versions.yml
"${task.process}":
    segemehl: \$(echo \$(segemehl.x 2>&1 | grep "ge5dee" | awk -F Z '{print substr(\$1, 2, 6)}' ))
END_VERSIONS
"""

NextFlow From line 23 of index/main.nf

"""
touch ${prefix}.idx

cat <<-END_VERSIONS > versions.yml
"${task.process}":
    segemehl: \$(echo \$(segemehl.x 2>&1 | grep "ge5dee" | awk -F Z '{print substr(\$1, 2, 6)}' ))
END_VERSIONS
"""

NextFlow From line 38 of index/main.nf

"""
STAR \\
    --genomeDir $index \\
    --readFilesIn $reads  \\
    --runThreadN $task.cpus \\
    --outFileNamePrefix $prefix. \\
    $out_sam_type \\
    $ignore_gtf \\
    $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")
    samtools: \$(echo \$(samtools --version 2>&1) | sed 's/^.*samtools //; s/Using.*\$//')
    gawk: \$(echo \$(gawk --version 2>&1) | sed 's/^.*GNU Awk //; s/, .*\$//')
END_VERSIONS
"""

NextFlow STAR From line 44 of align/main.nf

"""
touch ${prefix}Xd.out.bam
touch ${prefix}.Log.final.out
touch ${prefix}.Log.out
touch ${prefix}.Log.progress.out
touch ${prefix}.sortedByCoord.out.bam
touch ${prefix}.toTranscriptome.out.bam
touch ${prefix}.Aligned.unsort.out.bam
touch ${prefix}.unmapped_1.fastq.gz
touch ${prefix}.unmapped_2.fastq.gz
touch ${prefix}.tab
touch ${prefix}.Chimeric.out.junction
touch ${prefix}.out.sam

cat <<-END_VERSIONS > versions.yml
"${task.process}":
    star: \$(STAR --version | sed -e "s/STAR_//g")
    samtools: \$(echo \$(samtools --version 2>&1) | sed 's/^.*samtools //; s/Using.*\$//')
    gawk: \$(echo \$(gawk --version 2>&1) | sed 's/^.*GNU Awk //; s/, .*\$//')
END_VERSIONS
"""

NextFlow From line 76 of align/main.nf

"""
mkdir star
STAR \\
    --runMode genomeGenerate \\
    --genomeDir star/ \\
    --genomeFastaFiles $fasta \\
    --sjdbGTFfile $gtf \\
    --runThreadN $task.cpus \\
    $memory \\
    $args

cat <<-END_VERSIONS > versions.yml
"${task.process}":
    star: \$(STAR --version | sed -e "s/STAR_//g")
    samtools: \$(echo \$(samtools --version 2>&1) | sed 's/^.*samtools //; s/Using.*\$//')
    gawk: \$(echo \$(gawk --version 2>&1) | sed 's/^.*GNU Awk //; s/, .*\$//')
END_VERSIONS
"""

NextFlow STAR From line 26 of genomegenerate/main.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

cat <<-END_VERSIONS > versions.yml
"${task.process}":
    star: \$(STAR --version | sed -e "s/STAR_//g")
    samtools: \$(echo \$(samtools --version 2>&1) | sed 's/^.*samtools //; s/Using.*\$//')
    gawk: \$(echo \$(gawk --version 2>&1) | sed 's/^.*GNU Awk //; s/, .*\$//')
END_VERSIONS
"""

NextFlow SAMtools STAR From line 45 of genomegenerate/main.nf

"""
mkdir star
touch star/Genome
touch star/Log.out
touch star/SA
touch star/SAindex
touch star/chrLength.txt
touch star/chrName.txt
touch star/chrNameLength.txt
touch star/chrStart.txt
touch star/exonGeTrInfo.tab
touch star/exonInfo.tab
touch star/geneInfo.tab
touch star/genomeParameters.txt
touch star/sjdbInfo.txt
touch star/sjdbList.fromGTF.out.tab
touch star/sjdbList.out.tab
touch star/transcriptInfo.tab

cat <<-END_VERSIONS > versions.yml
"${task.process}":
    star: \$(STAR --version | sed -e "s/STAR_//g")
    samtools: \$(echo \$(samtools --version 2>&1) | sed 's/^.*samtools //; s/Using.*\$//')
    gawk: \$(echo \$(gawk --version 2>&1) | sed 's/^.*GNU Awk //; s/, .*\$//')
END_VERSIONS
"""

NextFlow From line 70 of genomegenerate/main.nf

"""
stringtie \\
    $bam \\
    $strandedness \\
    $reference \\
    -o ${prefix}.transcripts.gtf \\
    -A ${prefix}.gene.abundance.txt \\
    $coverage \\
    $ballgown \\
    -p $task.cpus \\
    $args

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

NextFlow StringTie From line 37 of stringtie/main.nf

"""
touch ${prefix}.transcripts.gtf
touch ${prefix}.gene.abundance.txt
touch ${prefix}.coverage.gtf
touch ${prefix}.ballgown

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

NextFlow From line 57 of stringtie/main.nf

"""
[ ! -f  ${prefix}.fastq.gz ] && ln -s $reads ${prefix}.fastq.gz
trim_galore \\
    $args \\
    --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 41 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
"""