Variant-calling pipeline for Restriction site-associated DNA sequencing

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Introduction

nf-core/radseq is a bioinformatics best-practice variant-calling pipeline for Restriction site-associated DNA sequencing (RADseq).

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

  1. Read QC ( FastQC )

  2. Adapter and quality trimming ( FastP )

  3. Choice of constructing psuedoreference (i) or using existing reference (ii)

    i. CDHIT-est -> Rainbow

    ii. No Preprossesing

  4. Choice of alignment software

    i. BWA

    ii. BWA-mem2

  5. UMI-based deduplicated ( UMI-tools )

  6. Index, merge and index alignments ( SAMtools )

  7. Construct intervals for freebayes ( BEDtools )

  8. Variant calling ( FreeBayes )

  9. Present QC for raw reads ( MultiQC )

Quick Start

  1. Install Nextflow ( >=21.10.3 )

  2. Install any of Docker , Singularity , Podman , Shifter or Charliecloud for full pipeline reproducibility (please only use Conda as a last resort; see docs )

  3. Download the pipeline and test it on a minimal dataset with a single command: 

    nextflow run nf-core/radseq -profile test,YOURPROFILE

    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 and are persistently observing issues downloading Singularity images directly due to timeout or network issues, then you can use the --singularity_pull_docker_container parameter to pull and convert the Docker image instead. Alternatively, you can 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.

  4. Start running your own analysis! 

    nextflow run nf-core/radseq -profile <docker/singularity/podman/shifter/charliecloud/conda/institute> --input samplesheet.csv

Documentation

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

Credits

dDocent was originally written by Jon Puritz and developed into nf-core/radseq by Gabriel Barrett

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 #radseq 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

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"""
bedops --merge ${bed.collect().join(" ")} \\
${args} \\
> ${prefix}.bed

cat <<-END_VERSIONS > versions.yml
"${task.process}":
    bedops: \$(bedops --version | awk '/version:/{print \$2}')
END_VERSIONS
"""
NextFlow Bedops From line 24 of merge/main.nf 
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"""
awk '{if (\$1 ~ /Cl/) clus = clus + 1; else  print \$3 "\\t" clus}' ${clstr} | \\
sed -e 's/[>dDocent_Contig_,...]//g' | \\
sort -g -k1 -S 2G > ${prefix}.sort.contig.cluster.ids

paste ${prefix}.sort.contig.cluster.ids ${totaluniqseq} > ${prefix}.contig.cluster.totaluniqseq

# cd-hit TO rainbow cluster format
sort -k2,2 -g ${prefix}.contig.cluster.totaluniqseq | \\
sed -e 's/NNNNNNNNNN/	/g' > ${prefix}.rclstr
cat <<-END_VERSIONS > versions.yml
"${task.process}":
    BusyBox: \$(busybox | sed -n -E 's/.*v([[:digit:].]+)\\s\\(.*/\\1/p')
    GNU: \$(sort --version | awk 'NR==1{print \$4}')
END_VERSIONS
"""
NextFlow cd-hit From line 26 of local/cdhit_to_rbdiv.nf 
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"""
awk '{if (\$1 ~ /Cl/) clus = clus + 1; else  print \$3 "\\t" clus}' ${clstr} | \\
sed -e 's/[>dDocent_Contig_,...]//g' | \\
sort -g -k1 > ${prefix}.sort.contig.cluster.ids

paste ${prefix}.sort.contig.cluster.ids <(awk '!/>/' ${totaluniqseq}) > ${prefix}.contig.cluster.Funiq

sed -e 's/NNNNNNNNNN/   /g' ${totaluniqseq} | \\
sort -k1 -S 2G | \\
awk '{print \$0 "\\t" NR}'  > ${prefix}.totaluniqseq.CN

join -t \$'\\t' -1 3 -2 1 ${prefix}.contig.cluster.Funiq ${prefix}.totaluniqseq.CN -o 2.3,1.2,2.1,2.2 > ${prefix}.contig.cluster.totaluniqseq

# cd-hit TO rainbow cluster format
sort -k2,2 -g ${prefix}.contig.cluster.totaluniqseq -S 2G | \\
sed -e 's/NNNNNNNNNN/	/g' > ${prefix}..rclstr

cat <<-END_VERSIONS > versions.yml
"${task.process}":
    BusyBox: \$(busybox | sed -n -E 's/.*v([[:digit:].]+)\\s\\(.*/\\1/p')
    GNU: \$(sort --version | awk 'NR==1{print \$4}')
END_VERSIONS
"""
NextFlow cd-hit From line 43 of local/cdhit_to_rbdiv.nf 
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"""
awk -v x="${withinIndv_MinDepth}" '(\$1 >= x)' *.uniq.seqs | \\
cut -f2 | \\
sed -e 's/NNNNNNNNNN/-/' >  total.uniqs

cut -f 1 -d "-" total.uniqs > total.u.F
cut -f 2 -d "-" total.uniqs > total.u.R

paste total.u.F total.u.R | \\
sort -k1 -S 2G > total.fr

awk -v x=${withinIndv_MinDepth} '\$1 >= x' *.uniq.seqs | \\
cut -f2 | \\
sed -e 's/NNNNNNNNNN/	/g' | \\
cut -f1 | \\
uniq | \\
sort -S 2G | \\
uniq -c > total.f.uniq

join -1 2 -2 1 -o 1.1,1.2,2.2 total.f.uniq total.fr | \\
awk '{print \$1 "\t" \$2 "NNNNNNNNNN" \$3}' | \\
awk -v x=${acrossIndv_MinDepth} '\$1 >= x' > uniq.k.${withinIndv_MinDepth}.c.${acrossIndv_MinDepth}.seqs

sort -k1 -r -n -S 2G uniq.k.${withinIndv_MinDepth}.c.${acrossIndv_MinDepth}.seqs | \\
cut -f2 > totaluniqseq
awk '{c= c + 1; print ">dDocent_Contig_" c "\\n" \$1}' totaluniqseq > ${prefix}_uniq.full.fasta

cat <<-END_VERSIONS > versions.yml
"${task.process}":
    BusyBox: \$(busybox | sed -n -E 's/.*v([[:digit:].]+)\\s\\(.*/\\1/p')
END_VERSIONS
"""
NextFlow From line 28 of local/combine_uniq_forward_reads.nf 
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"""
awk -v x=${withinIndv_MinDepth} '(\$1 >= x)' *.uniq.seqs | \\
cut -f2 | \\
perl -e 'while (<>) {chomp; \$z{\$_}++;} while((\$k,\$v) = each(%z)) {print "\$v\\t\$k\\n";}' | \\
awk -v x=${acrossIndv_MinDepth} '(\$1 >= x)' > uniq.k.${withinIndv_MinDepth}.c.${acrossIndv_MinDepth}.seqs

# order the sequences for reproducibility 
sort -k1 -r -n -S 2G uniq.k.${withinIndv_MinDepth}.c.${acrossIndv_MinDepth}.seqs | \\
cut -f2 > totaluniqseq 

awk '{c= c + 1; print ">dDocent_Contig_" c "\\n" \$1}' totaluniqseq > ${prefix}_uniq.full.fasta

cat <<-END_VERSIONS > versions.yml
"${task.process}":
    BusyBox: \$(busybox | sed -n -E 's/.*v([[:digit:].]+)\\s\\(.*/\\1/p')
    perl: \$(perl --version | sed -n -E '/^This is/ s/.*\\(v([[:digit:].]+)\\).*/\\1/p')
END_VERSIONS
"""
NextFlow From line 61 of local/combine_uniq_forward_reads.nf 
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"""
cat ${intersect} ${low_cov} > ${prefix}_cov.split.stats
echo "${lengths.join("\n")}" > ${prefix}_lengths.txt
MaxLen=\$(awk '{ print length() | "sort -rn" }' ${prefix}_lengths.txt| head -1)
MaxLen2=\$(( \$MaxLen / 2 ))

TT=\$(( \$MaxLen2 * 1000000 ))
DP=\$(awk '{print \$4}' ${cov} | sort -rn | perl -e '\$d=.001;@l=<>;print \$l[int(\$d*@l)]')
CC=\$( awk -v x=\$DP '\$4 < x' ${cov} | awk '{len=\$3-\$2;lc=len*\$4;tl=tl+lc} END {OFMT = "%.0f";print tl/"'${task.cpus}'"}')

awk -v x=\$DP '\$4 < x' ${prefix}_cov.split.stats | sort -k1,1 -k2,2 | awk -v cutoff=\$CC -v tt=\$TT 'BEGIN{i=1}
    {len=\$3-\$2;lc=len*\$4;cov = cov + lc
    if (NR == 1 && lc > tt) {x="mapped."i".bed";print \$1"\\t"\$2"\\t"\$3 > x; i=i+1; e=1}
    else if ( cov < cutoff && lc < tt) {x="mapped."i".bed";print \$1"\\t"\$2"\\t"\$3 > x; e=0}
    else if (lc > tt && e > 0 ) {x="mapped."i".bed"; print \$1"\\t"\$2"\\t"\$3 > x; cov=0;i=i+1; e=1}
    else if (lc > tt && e < 1 ) {i=i+1; x="mapped."i".bed"; print \$1"\\t"\$2"\\t"\$3 > x; cov=0;i=i+1;e=1}
    else if (cov > cutoff && lc < tt ) {i=i+1; x="mapped."i".bed"; print \$1"\\t"\$2"\\t"\$3 > x; cov=lc;e=0}
    }'
cat <<-END_VERSIONS > versions.yml
"${task.process}":
    BusyBox: \$(busybox | sed -n -E 's/.*v([[:digit:].]+)\\s\\(.*/\\1/p')
    perl: \$(perl --version | sed -n -E '/^This is/ s/.*\\(v([[:digit:].]+)\\).*/\\1/p')
END_VERSIONS
"""
NextFlow From line 24 of local/create_intervals.nf 
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"""
cat ${intersect} ${low_cov} > ${prefix}_cov.split.stats
echo "${lengths.join("\n")}" > ${prefix}_lengths.txt
MaxLen=\$(awk '{ print length() | "sort -rn" }' ${prefix}_lengths.txt| head -1)
MaxLen2=\$(( \$MaxLen / 2 ))

TT=\$(( \$MaxLen2 * 1000000 ))
DP=\$(awk '{print \$4}' ${cov} | sort -rn | perl -e '\$d=.00005;@l=<>;print \$l[int(\$d*@l)]')
CC=\$( awk -v x=\$DP '\$4 < x' ${cov} | awk '{len=\$3-\$2;lc=len*\$4;tl=tl+lc} END {OFMT = "%.0f";print tl/"'${task.cpus}'"}')

awk -v x=\$DP '\$4 < x' ${prefix}_cov.split.stats | sort -k1,1 -k2,2 | awk -v cutoff=\$CC -v tt=\$TT 'BEGIN{i=1}
    {len=\$3-\$2;lc=len*\$4;cov = cov + lc
    if (NR == 1 && lc > tt) {x="mapped."i".bed";print \$1"\\t"\$2"\\t"\$3 > x; i=i+1; e=1}
    else if ( cov < cutoff && lc < tt) {x="mapped."i".bed";print \$1"\\t"\$2"\\t"\$3 > x; e=0}
    else if (lc > tt && e > 0 ) {x="mapped."i".bed"; print \$1"\\t"\$2"\\t"\$3 > x; cov=0;i=i+1; e=1}
    else if (lc > tt && e < 1 ) {i=i+1; x="mapped."i".bed"; print \$1"\\t"\$2"\\t"\$3 > x; cov=0;i=i+1;e=1}
    else if (cov > cutoff && lc < tt ) {i=i+1; x="mapped."i".bed"; print \$1"\\t"\$2"\\t"\$3 > x; cov=lc;e=0}
    }'
cat <<-END_VERSIONS > versions.yml
"${task.process}":
    BusyBox: \$(busybox | sed -n -E 's/.*v([[:digit:].]+)\\s\\(.*/\\1/p')
    perl: \$(perl --version | sed -n -E '/^This is/ s/.*\\(v([[:digit:].]+)\\).*/\\1/p')
END_VERSIONS   
"""
NextFlow From line 49 of local/create_intervals.nf 
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"""
gunzip -c ${forward_reads} | \\
awk 'BEGIN{P=1}{if(P==1||P==2){gsub(/^[@]/,">");print}; if(P==4)P=0; P++}' | \\
awk '!/>/' > ${prefix}.forward

gunzip -c ${reverse_reads} | \\
awk 'BEGIN{P=1}{if(P==1||P==2){gsub(/^[@]/,">");print}; if(P==4)P=0; P++}' | \\
awk '!/>/' > ${prefix}.reverse

paste -d '-' ${prefix}.forward ${prefix}.reverse | \\
awk 'BEGIN{P=1}{if(P==1||P==2){gsub(/^[@]/,">");print}; if(P==4)P=0; P++}' | \\
sed -e 's/-/NNNNNNNNNN/' | \\
perl -e 'while (<>) {chomp; \$z{\$_}++;} while((\$k,\$v) = each(%z)) {print "\$v\\t\$k\\n";}' > ${prefix}.uniq.seqs

cat <<-END_VERSIONS > versions.yml
"${task.process}":
    BusyBox: \$(busybox | sed -n -E 's/.*v([[:digit:].]+)\\s\\(.*/\\1/p')
    perl: \$(perl --version | sed -n -E '/^This is/ s/.*\\(v([[:digit:].]+)\\).*/\\1/p')
END_VERSIONS
"""
NextFlow From line 26 of local/prepare_forward_reads.nf 
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        """
        gunzip -c ${forward_reads} | \\
        awk 'BEGIN{P=1}{if(P==1||P==2){gsub(/^[@]/,">");print}; if(P==4)P=0; P++}' | \\
        awk '!/>/' > ${prefix}.forward

        gunzip -c ${reverse_reads} | \\
        awk 'BEGIN{P=1}{if(P==1||P==2){gsub(/^[@]/,">");print}; if(P==4)P=0; P++}' | \\
        awk '!/>/' > ${prefix}.reverse

        paste ${prefix}.forward ${prefix}.reverse | sort -k1 -S 200M > ${prefix}.fr
		cut -f1 ${prefix}.fr | uniq -c > ${prefix}.f.uniq && cut -f2 ${prefix}.fr > ${prefix}.r
		awk '{for(i=0;i<\$1;i++)print}' ${prefix}.f.uniq > ${prefix}.f.uniq.e

        paste -d '-' ${prefix}.f.uniq.e ${prefix}.r | \\
        awk '!/NNN/'| \\
        sed -e 's/-/NNNNNNNNNN/' | \\
        sed -e 's/^[ \\t]*//' | \\
        sed -e 's/\\s/\\t/g' > ${prefix}.uniq.seqs

        rm ${prefix}.f.uniq.e ${prefix}.f.uniq ${prefix}.r ${prefix}.fr

cat <<-END_VERSIONS > versions.yml
"${task.process}":
    BusyBox: \$(busybox | sed -n -E 's/.*v([[:digit:].]+)\\s\\(.*/\\1/p')
END_VERSIONS
        """
NextFlow From line 49 of local/prepare_forward_reads.nf 
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"""
gunzip -c ${forward_reads} | \\
awk 'BEGIN{P=1}{if(P==1||P==2){gsub(/^[@]/,">");print}; if(P==4)P=0; P++}' | \\
awk '!/>/' | \\
perl -e 'while (<>) {chomp; \$z{\$_}++;} while((\$k,\$v) = each(%z)) {print "\$v\\t\$k\\n";}' > ${prefix}.uniq.seqs

cat <<-END_VERSIONS > versions.yml
"${task.process}":
    BusyBox: \$(busybox | sed -n -E 's/.*v([[:digit:].]+)\\s\\(.*/\\1/p')
    perl: \$(perl --version | sed -n -E '/^This is/ s/.*\\(v([[:digit:].]+)\\).*/\\1/p')
END_VERSIONS
"""
NextFlow From line 77 of local/prepare_forward_reads.nf 
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"""
awk 'BEGIN{P=1}{if(P==1||P==2){gsub(/^[@]/,">");print}; if(P==4)P=0; P++}' ${assembled_fastq} | \\
awk '!/>/' | \\
perl -e 'while (<>) {chomp; \$z{\$_}++;} while((\$k,\$v) = each(%z)) {print "\$v\\t\$k\\n";}' > ${prefix}.uniq.seqs

cat <<-END_VERSIONS > versions.yml
"${task.process}":
    BusyBox: \$(busybox | sed -n -E 's/.*v([[:digit:].]+)\\s\\(.*/\\1/p')
    perl: \$(perl --version | sed -n -E '/^This is/ s/.*\\(v([[:digit:].]+)\\).*/\\1/p')
END_VERSIONS
"""
NextFlow From line 90 of local/prepare_forward_reads.nf 
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"""
check_samplesheet.py \\
    $samplesheet \\
    samplesheet.valid.csv

cat <<-END_VERSIONS > versions.yml
"${task.process}":
    python: \$(python --version | sed 's/Python //g')
END_VERSIONS
"""
NextFlow From line 17 of local/samplesheet_check.nf 
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"""
bcftools concat \\
    --output ${prefix}.vcf.gz \\
    $args \\
    --threads $task.cpus \\
    ${vcfs}

cat <<-END_VERSIONS > versions.yml
"${task.process}":
    bcftools: \$(bcftools --version 2>&1 | head -n1 | sed 's/^.*bcftools //; s/ .*\$//')
END_VERSIONS
"""
NextFlow BCFtools From line 23 of concat/main.nf 
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"""
touch ${prefix}.vcf.gz

cat <<-END_VERSIONS > versions.yml
"${task.process}":
    bcftools: \$(bcftools --version 2>&1 | head -n1 | sed 's/^.*bcftools //; s/ .*\$//')
END_VERSIONS
"""
NextFlow From line 38 of concat/main.nf 
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"""
bcftools \\
    sort \\
    --output ${prefix}${interval}.vcf.gz \\
    $args \\
    $vcf

cat <<-END_VERSIONS > versions.yml
"${task.process}":
    bcftools: \$(bcftools --version 2>&1 | head -n1 | sed 's/^.*bcftools //; s/ .*\$//')
END_VERSIONS
"""
NextFlow BCFtools From line 24 of sort/main.nf 
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"""
touch ${prefix}.vcf.gz

cat <<-END_VERSIONS > versions.yml
"${task.process}":
    bcftools: \$(bcftools --version 2>&1 | head -n1 | sed 's/^.*bcftools //; s/ .*\$//')
END_VERSIONS
"""
NextFlow From line 40 of sort/main.nf 
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"""
bcftools stats \\
    $args \\
    $regions_file \\
    $targets_file \\
    $samples_file \\
    $vcf > ${prefix}.bcftools_stats.txt

cat <<-END_VERSIONS > versions.yml
"${task.process}":
    bcftools: \$(bcftools --version 2>&1 | head -n1 | sed 's/^.*bcftools //; s/ .*\$//')
END_VERSIONS
"""
NextFlow BCFtools From line 29 of stats/main.nf 
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"""
touch ${prefix}.bcftools_stats.txt

cat <<-END_VERSIONS > versions.yml
"${task.process}":
    bcftools: \$(bcftools --version 2>&1 | head -n1 | sed 's/^.*bcftools //; s/ .*\$//')
END_VERSIONS
"""
NextFlow From line 46 of stats/main.nf 
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"""
bedtools \\
    bamtobed \\
    $args \\
    -i $bam \\
    > ${prefix}.bed

cat <<-END_VERSIONS > versions.yml
"${task.process}":
    bedtools: \$(bedtools --version | sed -e "s/bedtools v//g")
END_VERSIONS
"""
NextFlow BEDTools From line 23 of bamtobed/main.nf 
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"""
bedtools \\
    coverage \\
    $args \\
    $reference \\
    -a $input_A \\
    -b $input_B \\
    > ${prefix}.cov

cat <<-END_VERSIONS > versions.yml
"${task.process}":
    bedtools: \$(echo \$(bedtools --version 2>&1) | sed 's/^.*bedtools v//' ))
END_VERSIONS
"""
NextFlow BEDTools From line 24 of coverage/main.nf 
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"""
bedtools \\
    intersect \\
    -a $intervals1 \\
    -b $intervals2 \\
    $args \\
    > ${prefix}.${extension}

cat <<-END_VERSIONS > versions.yml
"${task.process}":
    bedtools: \$(bedtools --version | sed -e "s/bedtools v//g")
END_VERSIONS
"""
NextFlow BEDTools From line 27 of intersect/main.nf 
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"""
touch ${prefix}.${extension}

cat <<-END_VERSIONS > versions.yml
"${task.process}":
    bedtools: \$(bedtools --version | sed -e "s/bedtools v//g")
END_VERSIONS
"""
NextFlow BEDTools From line 46 of intersect/main.nf 
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    """
    echo "${lengths.join("\n")}" > "${prefix}_lengths.txt"
    MaxLen=\$(awk '{ print length() | "sort -rn" }' "${prefix}_lengths.txt" | head -1)
    #split cov.stats file into high and low coverage intervals
	awk '\$4 > ${coverage_threshold}' ${regions} > ${prefix}_cov.high.stats
	awk '\$4 <= ${coverage_threshold}' ${regions} > ${prefix}_cov.low.stats
    MaxLen2=\$(("\$MaxLen" / 2))
    ML1=\$(("\$MaxLen2" + 1))
    bedtools \\
        makewindows \\
        -b ${prefix}_cov.high.stats \\
        -w \$MaxLen2 -s \$ML1 \\
        $args \\
        > ${prefix}.tab

cat <<-END_VERSIONS > versions.yml
"${task.process}":
    bedtools: \$(bedtools --version | sed -e "s/bedtools v//g")
END_VERSIONS
    """
NextFlow BEDTools From line 29 of makewindows/main.nf 
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"""
cat ${cov} | \\
bedtools sort -i - -faidx ${faidx} | \\
bedtools \\
    merge \\
    -i - \\
    $args \\
    > ${prefix}.cov
cat <<-END_VERSIONS > versions.yml
"${task.process}":
    bedtools: \$(bedtools --version | sed -e "s/bedtools v//g")
END_VERSIONS
"""
NextFlow BEDTools From line 25 of merge/main.nf 
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"""
bedtools \\
    sort \\
    -i $intervals \\
    $genome_cmd \\
    $args \\
    > ${prefix}_sorted.${extension}

cat <<-END_VERSIONS > versions.yml
"${task.process}":
    bedtools: \$(bedtools --version | sed -e "s/bedtools v//g")
END_VERSIONS
"""
NextFlow BEDTools From line 29 of sort/main.nf 
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"""
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 
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"""
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 
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"""
mkdir bwamem2
bwa-mem2 \\
    index \\
    $args \\
    $fasta -p bwamem2/${fasta}

cat <<-END_VERSIONS > versions.yml
"${task.process}":
    bwamem2: \$(echo \$(bwa-mem2 version 2>&1) | sed 's/.* //')
END_VERSIONS
"""
NextFlow Bwa-mem2 From line 22 of index/main.nf 
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"""
mkdir bwamem2
touch bwamem2/${fasta}.0123
touch bwamem2/${fasta}.ann
touch bwamem2/${fasta}.pac
touch bwamem2/${fasta}.amb
touch bwamem2/${fasta}.bwt.2bit.64

cat <<-END_VERSIONS > versions.yml
"${task.process}":
    bwamem2: \$(echo \$(bwa-mem2 version 2>&1) | sed 's/.* //')
END_VERSIONS
"""
NextFlow Bwa-mem2 From line 36 of index/main.nf 
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        """
        INDEX=`find -L ./ -name "*.amb" | sed 's/\\.amb\$//'`

        echo "${lengths.join("\n")}" > lengths.txt
        MLEN=\$(awk '{ print length() | "sort -rn" }' lengths.txt | head -1)
        INSERT=\$(( \$MLEN * 2 ))
        INSERTH=\$(( \$INSERT + 100 ))
        INSERTL=\$(( \$INSERT - 100 ))
        SD=\$(( \$INSERT / 5 ))

        bwa-mem2 \\
            mem \\
            $args \\
            -I \$INSERT,\$SD,\$INSERTH,\$INSERTL \\
            -R "@RG\\tID:${prefix}\\tSM:${prefix}\\tPL:Illumina" \\
            -t $task.cpus \\
            \$INDEX \\
            $reads \\
            | samtools view $args2 \\
            | samtools sort $args3 --threads $task.cpus -o ${prefix}.bam -

cat <<-END_VERSIONS > versions.yml
"${task.process}":
    bwamem2: \$(echo \$(bwa-mem2 version 2>&1) | sed 's/.* //')
    samtools: \$(echo \$(samtools --version 2>&1) | sed 's/^.*samtools //; s/Using.*\$//')
END_VERSIONS
        """
NextFlow SAMtools Bwa-mem2 From line 30 of mem/main.nf 
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        """
        INDEX=`find -L ./ -name "*.amb" | sed 's/\\.amb\$//'`
        bwa-mem2 \\
            mem \\
            $args \\
            -R "@RG\\tID:${prefix}\\tSM:${prefix}\\tPL:Illumina" \\
            -t $task.cpus \\
            \$INDEX \\
            $reads \\
            | samtools view $args2 \\
            | samtools sort $args3 --threads $task.cpus -o ${prefix}.bam -

cat <<-END_VERSIONS > versions.yml
"${task.process}":
    bwamem2: \$(echo \$(bwa-mem2 version 2>&1) | sed 's/.* //')
    samtools: \$(echo \$(samtools --version 2>&1) | sed 's/^.*samtools //; s/Using.*\$//')
END_VERSIONS
        """
NextFlow SAMtools Bwa-mem2 From line 58 of mem/main.nf 
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"""
touch ${prefix}.bam
cat <<-END_VERSIONS > versions.yml
"${task.process}":
    bwamem2: \$(echo \$(bwa-mem2 version 2>&1) | sed 's/.* //')
    samtools: \$(echo \$(samtools --version 2>&1) | sed 's/^.*samtools //; s/Using.*\$//')
END_VERSIONS
"""
NextFlow From line 79 of mem/main.nf 
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        """
        INDEX=`find -L ./ -name "*.amb" | sed 's/.amb//'`

        echo "${lengths.join("\n")}" > lengths.txt
        MLEN=\$(awk '{ print length() | "sort -rn" }' lengths.txt | head -1)
        INSERT=\$(( \$MLEN * 2 ))
        INSERTH=\$(( \$INSERT + 100 ))
        INSERTL=\$(( \$INSERT - 100 ))
        SD=\$(( \$INSERT / 5 ))

        bwa mem \\
            $args \\
            -I \$INSERT,\$SD,\$INSERTH,\$INSERTL \\
            -R "@RG\\tID:${prefix}\\tSM:${prefix}\\tPL:Illumina" \\
            -t $task.cpus \\
            \$INDEX \\
            $reads \\
            | samtools view $args2 \\
            | samtools sort $args3 --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 30 of mem/main.nf 
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        """
        INDEX=`find -L ./ -name "*.amb" | sed 's/.amb//'`

        bwa mem \\
            $args \\
            -R "@RG\\tID:${prefix}\\tSM:${prefix}\\tPL:Illumina" \\
            -t $task.cpus \\
            \$INDEX \\
            $reads \\
            | samtools view $args2 \\
            | samtools sort $args3 --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 57 of mem/main.nf 
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"""
[ ! -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 \\
    $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 40 of fastp/main.nf 
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"""
MaxLen="\$(awk '!/>/' ${uniq_full_fasta}  | \\
    awk '(NR==1||length<shortest){shortest=length} END {print shortest}')"

fastp \\
    --in1 ${reads} \\
    --out1 ${prefix}.fastp.fastq.gz \\
    --thread ${task.cpus} \\
    ${args} \\
    --disable_quality_filtering \\
    --length_required \$MaxLen \\
    &> fastp.log

# Fastq back to Fasta
gunzip ${prefix}.fastp.fastq.gz
awk 'BEGIN{P=1}{if(P==1||P==2){gsub(/^[@]/,">");print}; if(P==4)P=0; P++}' ${prefix}.fastp.fastq | \\
    paste - - | \\
    sort -k1,1 -V | \\
    tr "\\t" "\\n" > ${prefix}.uniq.fasta

awk '!/>/' ${prefix}.uniq.fasta > ${prefix}.totaluniqseq

cat <<-END_VERSIONS > versions.yml
"${task.process}":
    fastp: \$(fastp --version 2>&1 | sed -e "s/fastp //g")
    BusyBox: \$(busybox | sed -n -E 's/.*v([[:digit:].]+)\\s\\(.*/\\1/p')
END_VERSIONS
"""
NextFlow fastp From line 58 of fastp/main.nf 
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"""
MaxLen="\$(awk '!/>/' ${uniq_full_fasta}  | \\
    awk '(NR==1||length<shortest){shortest=length} END {print shortest}')"

fastp \\
    --in1 ${reads} \\
    --out1 ${prefix}.fastp.fastq.gz \\
    --thread ${task.cpus} \\
    ${args} \\
    --disable_quality_filtering \\
    --length_required \$MaxLen \\
    &> fastp.log

# Fastq back to Fasta
gunzip ${prefix}.fastp.fastq.gz
awk 'BEGIN{P=1}{if(P==1||P==2){gsub(/^[@]/,">");print}; if(P==4)P=0; P++}' ${prefix}.fastp.fastq | \\
    paste - - | \\
    sort -k1,1 -V | \\
    tr "\\t" "\\n" > ${prefix}.uniq.fasta

awk '!/>/' ${prefix}.uniq.fasta > ${prefix}.totaluniqseq

cat <<-END_VERSIONS > versions.yml
"${task.process}":
    fastp: \$(fastp --version 2>&1 | sed -e "s/fastp //g")
    BusyBox: \$(busybox | sed -n -E 's/.*v([[:digit:].]+)\\s\\(.*/\\1/p')
END_VERSIONS
"""
NextFlow fastp From line 87 of fastp/main.nf 
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"""
[ ! -f  ${prefix}.fastq.gz ] && ln -sf ${reads} ${prefix}.fastq.gz
fastp \\
    --stdout \\
    --in1 ${prefix}.fastq.gz \\
    --out1  ${prefix}.fastp.fastq.gz \\
    --thread $task.cpus \\
    --json ${prefix}.fastp.json \\
    --html ${prefix}.fastp.html \\
    $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 116 of fastp/main.nf 
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"""
[ ! -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 \\
    $fail_fastq \\
    $merge_fastq \\
    --thread $task.cpus \\
    $args \\
    $umi_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 136 of fastp/main.nf 
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"""
[ ! -f  ${prefix}.fastq.gz ] && ln -sf $reads ${prefix}.fastq.gz
fastp \\
    --stdout \\
    --in1 ${prefix}.fastq.gz \\
    --out1  ${prefix}.fastp.fastq.gz \\
    --thread $task.cpus \\
    --json ${prefix}.fastp.json \\
    --html ${prefix}.fastp.html \\
    $fail_fastq \\
    $umi_args \\
    $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 159 of fastp/main.nf 
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"""
[ ! -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 \\
    $fail_fastq \\
    $merge_fastq \\
    --thread $task.cpus \\
    $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 179 of fastp/main.nf 
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"""
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 
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"""
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 
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"""
freebayes \\
    -f $fasta \\
    $targets_file \\
    $samples_file \\
    $populations_file \\
    $cnv_file \\
    $args \\
    $bam | \\
bgzip -c > ${prefix}${interval}.vcf.gz
cat <<-END_VERSIONS > versions.yml
"${task.process}":
    freebayes: \$(echo \$(freebayes --version 2>&1) | sed 's/version:\s*v//g' )
END_VERSIONS
"""
NextFlow FreeBayes From line 34 of freebayes/main.nf 
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"""
minimap2 \\
    $args \\
    -t $task.cpus \\
    "${reference ?: reads}" \\
    "$reads" \\
    $cigar_paf \\
    $set_cigar_bam \\
    $bam_output


cat <<-END_VERSIONS > versions.yml
"${task.process}":
    minimap2: \$(minimap2 --version 2>&1)
END_VERSIONS
"""
NextFlow Minimap2 From line 32 of align/main.nf 
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"""
minimap2 \\
    -t $task.cpus \\
    -d ${fasta.baseName}.mmi \\
    $args \\
    $fasta

cat <<-END_VERSIONS > versions.yml
"${task.process}":
    minimap2: \$(minimap2 --version 2>&1)
END_VERSIONS
"""
NextFlow Minimap2 From line 22 of index/main.nf 
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"""
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 
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"""
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 
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"""
samtools \\
    faidx \\
    $args \\
    $fasta

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 faidx/main.nf 
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"""
touch ${fasta}.fai
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 faidx/main.nf 
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"""
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 
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"""
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 
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"""
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 
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"""
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 
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"""
samtools \\
    merge \\
    --threads ${task.cpus-1} \\
    $args \\
    ${reference} \\
    ${prefix}.${file_type} \\
    $input_files

cat <<-END_VERSIONS > versions.yml
"${task.process}":
    samtools: \$(echo \$(samtools --version 2>&1) | sed 's/^.*samtools //; s/Using.*\$//')
END_VERSIONS
"""
NextFlow SAMtools From line 30 of merge/main.nf 
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"""
touch ${prefix}.${file_type}

cat <<-END_VERSIONS > versions.yml
"${task.process}":
    samtools: \$(echo \$(samtools --version 2>&1) | sed 's/^.*samtools //; s/Using.*\$//')
END_VERSIONS
"""
NextFlow From line 48 of merge/main.nf 
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"""
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 
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"""
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 
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"""
seqtk \\
    seq \\
    $args \\
    $fastx | \\
    gzip -c > ${prefix}.seqtk-seq.${extension}.gz

cat <<-END_VERSIONS > versions.yml
"${task.process}":
    seqtk: \$(echo \$(seqtk 2>&1) | sed 's/^.*Version: //; s/ .*\$//')
END_VERSIONS
"""
NextFlow seqtk From line 28 of seq/main.nf 
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"""
tabix $args $tab

cat <<-END_VERSIONS > versions.yml
"${task.process}":
    tabix: \$(echo \$(tabix -h 2>&1) | sed 's/^.*Version: //; s/ .*\$//')
END_VERSIONS
"""
NextFlow tabix From line 23 of tabix/main.nf 
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"""
touch ${tab}.tbi
cat <<-END_VERSIONS > versions.yml

"${task.process}":
    tabix: \$(echo \$(tabix -h 2>&1) | sed 's/^.*Version: //; s/ .*\$//')
END_VERSIONS
"""
NextFlow From line 34 of tabix/main.nf 
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"""
PYTHONHASHSEED=0 umi_tools \\
    dedup \\
    -I $bam \\
    -S ${prefix}_dedup.bam \\
    $stats \\
    $paired \\
    $args

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 31 of dedup/main.nf
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Created: 1yr ago
Updated: 1yr ago
Maitainers: public
URL: https://nf-co.re/radseq
Name: radseq
Version: dev
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