A fully reproducible and state-of-the-art ancient DNA analysis pipeline

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A fully reproducible and state-of-the-art ancient DNA analysis pipeline .

Introduction

nf-core/eager is a scalable and reproducible bioinformatics best-practise processing pipeline for genomic NGS sequencing data, with a focus on ancient DNA (aDNA) data. It is ideal for the (palaeo)genomic analysis of humans, animals, plants, microbes and even microbiomes.

The pipeline is built using Nextflow , a workflow tool to run tasks across multiple compute infrastructures in a very portable manner. It comes with docker containers making installation trivial and results highly reproducible. The pipeline pre-processes raw data from FASTQ inputs, or preprocessed BAM inputs. It can align reads and performs extensive general NGS and aDNA specific quality-control on the results. It comes with docker, singularity or conda containers making installation trivial and results highly reproducible.

nf-core/eager schematic workflow

Quick Start

  1. Install nextflow ( >=20.07.1 )

  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/eager -profile test,<docker/singularity/podman/shifter/charliecloud/conda/institute>

    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.

  4. Start running your own analysis! 

    nextflow run nf-core/eager -profile <docker/singularity/podman/conda/institute> --input '*_R{1,2}.fastq.gz' --fasta '<your_reference>.fasta'

  5. Once your run has completed successfully, clean up the intermediate files. 

    nextflow clean -f -k

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

N.B. You can see an overview of the run in the MultiQC report located at ./results/MultiQC/multiqc_report.html

Modifications to the default pipeline are easily made using various options as described in the documentation.

Pipeline Summary

Default Steps

By default the pipeline currently performs the following:

  • Create reference genome indices for mapping ( bwa , samtools , and picard )

  • Sequencing quality control ( FastQC )

  • Sequencing adapter removal, paired-end data merging ( AdapterRemoval )

  • Read mapping to reference using ( bwa aln , bwa mem , CircularMapper , or bowtie2 )

  • Post-mapping processing, statistics and conversion to bam ( samtools )

  • Ancient DNA C-to-T damage pattern visualisation ( DamageProfiler )

  • PCR duplicate removal ( DeDup or MarkDuplicates )

  • Post-mapping statistics and BAM quality control ( Qualimap )

  • Library Complexity Estimation ( preseq )

  • Overall pipeline statistics summaries ( MultiQC )

Additional Steps

Additional functionality contained by the pipeline currently includes:

Input

  • Automatic merging of complex sequencing setups (e.g. multiple lanes, sequencing configurations, library types)

Preprocessing

  • Illumina two-coloured sequencer poly-G tail removal ( fastp )

  • Post-AdapterRemoval trimming of FASTQ files prior mapping ( fastp )

  • Automatic conversion of unmapped reads to FASTQ ( samtools )

  • Host DNA (mapped reads) stripping from input FASTQ files (for sensitive samples)

aDNA Damage manipulation

  • Damage removal/clipping for UDG+/UDG-half treatment protocols ( BamUtil )

  • Damaged reads extraction and assessment ( PMDTools )

  • Nuclear DNA contamination estimation of human samples ( angsd )

Genotyping

  • Creation of VCF genotyping files ( GATK UnifiedGenotyper , GATK HaplotypeCaller and FreeBayes )

  • Creation of EIGENSTRAT genotyping files ( pileupCaller )

  • Creation of Genotype Likelihood files ( angsd )

  • Consensus sequence FASTA creation ( VCF2Genome )

  • SNP Table generation ( MultiVCFAnalyzer )

Biological Information

  • Mitochondrial to Nuclear read ratio calculation ( MtNucRatioCalculator )

  • Statistical sex determination of human individuals ( Sex.DetERRmine )

Metagenomic Screening

  • Low-sequenced complexity filtering ( BBduk )

  • Taxonomic binner with alignment ( MALT )

  • Taxonomic binner without alignment ( Kraken2 )

  • aDNA characteristic screening of taxonomically binned data from MALT ( MaltExtract )

Functionality Overview

A graphical overview of suggested routes through the pipeline depending on context can be seen below.

nf-core/eager metro map

Documentation

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

  1. Nextflow installation

  2. Pipeline configuration

  3. Running the pipeline

    • This includes tutorials, FAQs, and troubleshooting instructions

  4. Output and how to interpret the results

Credits

This pipeline was mostly written by Alexander Peltzer ( apeltzer ) and James A. Fellows Yates , with contributions from Stephen Clayton , Thiseas C. Lamnidis , Maxime Borry , Zandra Fagernäs , Aida Andrades Valtueña and Maxime Garcia and the nf-core community.

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

Authors (alphabetical)

Additional Contributors (alphabetical)

Those who have provided conceptual guidance, suggestions, bug reports etc.

If you've contributed and you're missing in here, please let us know and we will add you in of course!

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

Citations

If you use nf-core/eager for your analysis, please cite the eager preprint as follows:

Fellows Yates JA, Lamnidis TC, Borry M, Valtueña Andrades A, Fagernäs Z, Clayton S, Garcia MU, Neukamm J, Peltzer A. 2021. Reproducible, portable, and efficient ancient genome reconstruction with nf-core/eager. PeerJ 9:e10947. DOI: 10.7717/peerj.10947 .

You can cite the eager zenodo record for a specific version using the following doi: 10.5281/zenodo.3698082

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 .

In addition, references of tools and data used in this pipeline are as follows:

Data References

This repository uses test data from the following studies:

  • Fellows Yates, J. A. et al. (2017) ‘Central European Woolly Mammoth Population Dynamics: Insights from Late Pleistocene Mitochondrial Genomes’, Scientific reports, 7(1), p. 17714. doi: 10.1038/s41598-017-17723-1 .

  • Gamba, C. et al. (2014) ‘Genome flux and stasis in a five millennium transect of European prehistory’, Nature communications, 5, p. 5257. doi: 10.1038/ncomms6257 .

  • Star, B. et al. (2017) ‘Ancient DNA reveals the Arctic origin of Viking Age cod from Haithabu, Germany’, Proceedings of the National Academy of Sciences of the United States of America, 114(34), pp. 9152–9157. doi: 10.1073/pnas.1710186114 .

  • de Barros Damgaard, P. et al. (2018). '137 ancient human genomes from across the Eurasian steppes.', Nature, 557(7705), 369–374. doi: 10.1038/s41586-018-0094-2

Code Snippets

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"""
pigz -f -d -p ${task.cpus} $zipped_fasta
"""
NextFlow From line 193 of master/main.nf 
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"""
bwa index $fasta
mkdir BWAIndex && mv ${fasta}* BWAIndex
"""
NextFlow BWA From line 504 of master/main.nf 
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"""
bowtie2-build --threads ${task.cpus} $fasta $fasta
mkdir BT2Index && mv ${fasta}* BT2Index
"""
NextFlow Bowtie 2 From line 533 of master/main.nf 
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"""
samtools faidx $fasta
"""
NextFlow SAMtools From line 575 of master/main.nf 
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"""
picard -Xmx${task.memory.toMega()}M CreateSequenceDictionary R=$fasta O="${fasta.baseName}.dict"
"""
NextFlow Picard From line 615 of master/main.nf 
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"""
samtools fastq -t ${bam} | pigz -p ${task.cpus} > ${base}.converted.fastq.gz
"""
NextFlow SAMtools From line 643 of master/main.nf 
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"""
samtools index ${bam} ${size}
"""
NextFlow SAMtools From line 664 of master/main.nf 
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"""
fastqc -t ${task.cpus} -q $r1 $r2
rename 's/_fastqc\\.zip\$/_raw_fastqc.zip/' *_fastqc.zip
rename 's/_fastqc\\.html\$/_raw_fastqc.html/' *_fastqc.html
"""
NextFlow FastQC From line 699 of master/main.nf 
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"""
fastqc -t ${task.cpus} -q $r1
rename 's/_fastqc\\.zip\$/_raw_fastqc.zip/' *_fastqc.zip
rename 's/_fastqc\\.html\$/_raw_fastqc.html/' *_fastqc.html
"""
NextFlow FastQC From line 705 of master/main.nf 
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"""
fastp --in1 ${r1} --out1 "${r1.baseName}.pG.fq.gz" -A -g --poly_g_min_len "${params.complexity_filter_poly_g_min}" -Q -L -w ${task.cpus} --json "${r1.baseName}"_L${lane}_fastp.json 
"""
NextFlow fastp From line 746 of master/main.nf 
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"""
fastp --in1 ${r1} --in2 ${r2} --out1 "${r1.baseName}.pG.fq.gz" --out2 "${r2.baseName}.pG.fq.gz" -A -g --poly_g_min_len "${params.complexity_filter_poly_g_min}" -Q -L -w ${task.cpus} --json "${libraryid}"_L${lane}_polyg_fastp.json 
"""
NextFlow fastp From line 750 of master/main.nf 
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"""
mkdir -p output

AdapterRemoval --file1 ${r1} --file2 ${r2} --basename ${base}.pe --gzip --threads ${task.cpus} --qualitymax ${params.qualitymax} --collapse ${preserve5p} --trimns --trimqualities ${adapters_to_remove} --minlength ${params.clip_readlength} --minquality ${params.clip_min_read_quality} --minadapteroverlap ${params.min_adap_overlap}

cat *.collapsed.gz *.collapsed.truncated.gz *.singleton.truncated.gz *.pair1.truncated.gz *.pair2.truncated.gz > output/${base}.pe.combined.tmp.fq.gz

mv *.settings output/

## Add R_ and L_ for unmerged reads for DeDup compatibility
AdapterRemovalFixPrefix -Xmx${task.memory.toGiga()}g output/${base}.pe.combined.tmp.fq.gz | pigz -p ${task.cpus - 1} > output/${base}.pe.combined.fq.gz

"""
NextFlow AdapterRemoval From line 820 of master/main.nf 
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"""
mkdir -p output

AdapterRemoval --file1 ${r1} --file2 ${r2} --basename ${base}.pe --gzip --threads ${task.cpus} --qualitymax ${params.qualitymax} --collapse ${preserve5p} --trimns --trimqualities ${adapters_to_remove} --minlength ${params.clip_readlength} --minquality ${params.clip_min_read_quality} --minadapteroverlap ${params.min_adap_overlap}

cat *.collapsed.gz *.singleton.truncated.gz *.pair1.truncated.gz *.pair2.truncated.gz > output/${base}.pe.combined.tmp.fq.gz

mv *.settings output/

## Add R_ and L_ for unmerged reads for DeDup compatibility
AdapterRemovalFixPrefix -Xmx${task.memory.toGiga()}g output/${base}.pe.combined.tmp.fq.gz | pigz -p ${task.cpus - 1} > output/${base}.pe.combined.fq.gz

"""
NextFlow AdapterRemoval From line 835 of master/main.nf 
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"""
mkdir -p output
AdapterRemoval --file1 ${r1} --file2 ${r2} --basename ${base}.pe  --gzip --threads ${task.cpus} --qualitymax ${params.qualitymax} --collapse ${preserve5p} --trimns --trimqualities ${adapters_to_remove} --minlength ${params.clip_readlength} --minquality ${params.clip_min_read_quality} --minadapteroverlap ${params.min_adap_overlap}

cat *.collapsed.gz *.collapsed.truncated.gz > output/${base}.pe.combined.tmp.fq.gz

## Add R_ and L_ for unmerged reads for DeDup compatibility
AdapterRemovalFixPrefix -Xmx${task.memory.toGiga()}g output/${base}.pe.combined.tmp.fq.gz | pigz -p ${task.cpus - 1} > output/${base}.pe.combined.fq.gz

mv *.settings output/
"""
NextFlow AdapterRemoval From line 850 of master/main.nf 
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"""
mkdir -p output
AdapterRemoval --file1 ${r1} --file2 ${r2} --basename ${base}.pe  --gzip --threads ${task.cpus} --qualitymax ${params.qualitymax} --collapse ${preserve5p} --trimns --trimqualities ${adapters_to_remove} --minlength ${params.clip_readlength} --minquality ${params.clip_min_read_quality} --minadapteroverlap ${params.min_adap_overlap}

cat *.collapsed.gz > output/${base}.pe.combined.tmp.fq.gz

## Add R_ and L_ for unmerged reads for DeDup compatibility
AdapterRemovalFixPrefix -Xmx${task.memory.toGiga()}g output/${base}.pe.combined.tmp.fq.gz | pigz -p ${task.cpus - 1} > output/${base}.pe.combined.fq.gz

mv *.settings output/
"""
NextFlow AdapterRemoval From line 863 of master/main.nf 
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"""
mkdir -p output
AdapterRemoval --file1 ${r1} --file2 ${r2} --basename ${base}.pe --gzip --threads ${task.cpus} --qualitymax ${params.qualitymax} --collapse ${preserve5p} --adapter1 "" --adapter2 ""

cat *.collapsed.gz *.pair1.truncated.gz *.pair2.truncated.gz > output/${base}.pe.combined.tmp.fq.gz

## Add R_ and L_ for unmerged reads for DeDup compatibility
AdapterRemovalFixPrefix -Xmx${task.memory.toGiga()}g output/${base}.pe.combined.tmp.fq.gz | pigz -p ${task.cpus - 1} > output/${base}.pe.combined.fq.gz

mv *.settings output/
"""
NextFlow AdapterRemoval From line 877 of master/main.nf 
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"""
mkdir -p output
AdapterRemoval --file1 ${r1} --file2 ${r2} --basename ${base}.pe --gzip --threads ${task.cpus} --qualitymax ${params.qualitymax} --collapse ${preserve5p}  --adapter1 "" --adapter2 ""

cat *.collapsed.gz > output/${base}.pe.combined.tmp.fq.gz

## Add R_ and L_ for unmerged reads for DeDup compatibility
AdapterRemovalFixPrefix -Xmx${task.memory.toGiga()}g output/${base}.pe.combined.tmp.fq.gz | pigz -p ${task.cpus - 1} > output/${base}.pe.combined.fq.gz

mv *.settings output/
"""
NextFlow AdapterRemoval From line 891 of master/main.nf 
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"""
mkdir -p output
AdapterRemoval --file1 ${r1} --file2 ${r2} --basename ${base}.pe --gzip --threads ${task.cpus} --qualitymax ${params.qualitymax} ${preserve5p} --trimns --trimqualities ${adapters_to_remove} --minlength ${params.clip_readlength} --minquality ${params.clip_min_read_quality} --minadapteroverlap ${params.min_adap_overlap}

mv ${base}.pe.pair*.truncated.gz *.settings output/
"""
NextFlow AdapterRemoval From line 904 of master/main.nf 
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"""
mkdir -p output
AdapterRemoval --file1 ${r1} --basename ${base}.se --gzip --threads ${task.cpus} --qualitymax ${params.qualitymax} ${preserve5p} --trimns --trimqualities ${adapters_to_remove} --minlength ${params.clip_readlength} --minquality ${params.clip_min_read_quality} --minadapteroverlap ${params.min_adap_overlap}
mv *.settings *.se.truncated.gz output/
"""
NextFlow AdapterRemoval From line 912 of master/main.nf 
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"""
mkdir -p output
AdapterRemoval --file1 ${r1} --basename ${base}.se --gzip --threads ${task.cpus} --qualitymax ${params.qualitymax} ${preserve5p} --adapter1 "" --adapter2 ""
mv *.settings *.se.truncated.gz output/
"""
NextFlow AdapterRemoval From line 919 of master/main.nf 
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"""
fastp --in1 ${r1} --trim_front1 ${params.post_ar_trim_front} --trim_tail1 ${params.post_ar_trim_tail} -A -G -Q -L -w ${task.cpus} --out1 "${libraryid}"_L"${lane}"_R1_postartrimmed.fq.gz
"""
NextFlow fastp From line 996 of master/main.nf 
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"""
fastp --in1 ${r1} --in2 ${r2}  --trim_front1 ${params.post_ar_trim_front} --trim_tail1 ${params.post_ar_trim_tail} --trim_front2 ${params.post_ar_trim_front2} --trim_tail2 ${params.post_ar_trim_tail2} -A -G -Q -L -w ${task.cpus} --out1 "${libraryid}"_L"${lane}"_R1_postartrimmed.fq.gz --out2 "${libraryid}"_L"${lane}"_R2_postartrimmed.fq.gz
"""
NextFlow fastp From line 1000 of master/main.nf 
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"""
cat ${r1} > "${libraryid}"_R1_lanemerged.fq.gz
cat ${r2} > "${libraryid}"_R2_lanemerged.fq.gz
"""
NextFlow From line 1132 of master/main.nf 
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"""
cat ${r1} > "${libraryid}"_R1_lanemerged.fq.gz
"""
NextFlow From line 1137 of master/main.nf 
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"""
cat ${r1} > "${libraryid}"_R1_lanemerged.fq.gz
cat ${r2} > "${libraryid}"_R2_lanemerged.fq.gz
"""
NextFlow From line 1205 of master/main.nf 
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"""
cat ${r1} > "${libraryid}"_R1_lanemerged.fq.gz
"""
NextFlow From line 1210 of master/main.nf 
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"""
fastqc -t ${task.cpus} -q ${r1} ${r2}
"""
NextFlow FastQC From line 1238 of master/main.nf 
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"""
fastqc -t ${task.cpus} -q ${r1}
"""
NextFlow FastQC From line 1242 of master/main.nf 
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"""
bwa aln -t ${task.cpus} $fasta ${r1} -n ${params.bwaalnn} -l ${params.bwaalnl} -k ${params.bwaalnk} -o ${params.bwaalno} -f ${libraryid}.r1.sai
bwa aln -t ${task.cpus} $fasta ${r2} -n ${params.bwaalnn} -l ${params.bwaalnl} -k ${params.bwaalnk} -o ${params.bwaalno} -f ${libraryid}.r2.sai
bwa sampe -r "@RG\\tID:ILLUMINA-${libraryid}\\tSM:${samplename}\\tPL:illumina\\tPU:ILLUMINA-${libraryid}-${seqtype}" $fasta ${libraryid}.r1.sai ${libraryid}.r2.sai ${r1} ${r2} | samtools sort -@ ${task.cpus - 1} -O bam - > ${libraryid}_"${seqtype}".mapped.bam
samtools index "${libraryid}"_"${seqtype}".mapped.bam ${size}
"""
NextFlow SAMtools BWA From line 1276 of master/main.nf 
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"""
bwa aln -t ${task.cpus} ${fasta} ${r1} -n ${params.bwaalnn} -l ${params.bwaalnl} -k ${params.bwaalnk} -o ${params.bwaalno} -f ${libraryid}.sai
bwa samse -r "@RG\\tID:ILLUMINA-${libraryid}\\tSM:${samplename}\\tPL:illumina\\tPU:ILLUMINA-${libraryid}-${seqtype}" $fasta ${libraryid}.sai $r1 | samtools sort -@ ${task.cpus - 1} -O bam - > "${libraryid}"_"${seqtype}".mapped.bam
samtools index "${libraryid}"_"${seqtype}".mapped.bam ${size}
"""
NextFlow SAMtools BWA From line 1284 of master/main.nf 
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"""
bwa mem -t ${split_cpus} $fasta $r1 $r2 -R "@RG\\tID:ILLUMINA-${libraryid}\\tSM:${samplename}\\tPL:illumina\\tPU:ILLUMINA-${libraryid}-${seqtype}" | samtools sort -@ ${split_cpus} -O bam - > "${libraryid}"_"${seqtype}".mapped.bam
samtools index ${size} -@ ${task.cpus} "${libraryid}"_"${seqtype}".mapped.bam
"""
NextFlow SAMtools BWA From line 1316 of master/main.nf 
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"""
bwa mem -t ${split_cpus} $fasta $r1 -R "@RG\\tID:ILLUMINA-${libraryid}\\tSM:${samplename}\\tPL:illumina\\tPU:ILLUMINA-${libraryid}-${seqtype}" | samtools sort -@ ${split_cpus} -O bam - > "${libraryid}"_"${seqtype}".mapped.bam
samtools index -@ ${task.cpus} "${libraryid}"_"${seqtype}".mapped.bam ${size} 
"""
NextFlow SAMtools BWA From line 1321 of master/main.nf 
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"""
circulargenerator -Xmx${task.memory.toGiga()}g -e ${params.circularextension} -i $fasta -s ${params.circulartarget}
bwa index $prefix
"""
NextFlow BWA From line 1352 of master/main.nf 
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"""
bwa aln -t ${task.cpus} $elongated_root $r1 -n ${params.bwaalnn} -l ${params.bwaalnl} -k ${params.bwaalnk} -f ${libraryid}.r1.sai
bwa aln -t ${task.cpus} $elongated_root $r2 -n ${params.bwaalnn} -l ${params.bwaalnl} -k ${params.bwaalnk} -f ${libraryid}.r2.sai
bwa sampe -r "@RG\\tID:ILLUMINA-${libraryid}\\tSM:${samplename}\\tPL:illumina\\tPU:ILLUMINA-${libraryid}-${seqtype}" $elongated_root ${libraryid}.r1.sai ${libraryid}.r2.sai $r1 $r2 > tmp.out
realignsamfile -Xmx${task.memory.toGiga()}g -e ${params.circularextension} -i tmp.out -r $fasta $filter 
samtools sort -@ ${task.cpus} -O bam tmp_realigned.bam > ${libraryid}_"${seqtype}".mapped.bam
samtools index "${libraryid}"_"${seqtype}".mapped.bam ${size} 
"""
NextFlow SAMtools BWA From line 1382 of master/main.nf 
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""" 
bwa aln -t ${task.cpus} $elongated_root $r1 -n ${params.bwaalnn} -l ${params.bwaalnl} -k ${params.bwaalnk} -f ${libraryid}.sai
bwa samse -r "@RG\\tID:ILLUMINA-${libraryid}\\tSM:${samplename}\\tPL:illumina\\tPU:ILLUMINA-${libraryid}-${seqtype}" $elongated_root ${libraryid}.sai $r1 > tmp.out
realignsamfile -Xmx${task.memory.toGiga()}g -e ${params.circularextension} -i tmp.out -r $fasta $filter 
samtools sort -@ ${task.cpus} -O bam tmp_realigned.bam > "${libraryid}"_"${seqtype}".mapped.bam
samtools index "${libraryid}"_"${seqtype}".mapped.bam ${size}
"""
NextFlow SAMtools BWA From line 1391 of master/main.nf 
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"""
bowtie2 -x ${fasta} -1 ${r1} -2 ${r2} -p ${split_cpus} ${sensitivity} ${bt2n} ${bt2l} ${trim5} ${trim3} --maxins ${params.bt2_maxins} --rg-id ILLUMINA-${libraryid} --rg SM:${samplename} --rg PL:illumina --rg PU:ILLUMINA-${libraryid}-${seqtype} 2> "${libraryid}"_bt2.log | samtools sort -@ ${split_cpus} -O bam > "${libraryid}"_"${seqtype}".mapped.bam
samtools index "${libraryid}"_"${seqtype}".mapped.bam ${size}
"""
NextFlow SAMtools Bowtie 2 From line 1462 of master/main.nf 
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"""
bowtie2 -x ${fasta} -U ${r1} -p ${split_cpus} ${sensitivity} ${bt2n} ${bt2l} ${trim5} ${trim3} --rg-id ILLUMINA-${libraryid} --rg SM:${samplename} --rg PL:illumina --rg PU:ILLUMINA-${libraryid}-${seqtype} 2> "${libraryid}"_bt2.log | samtools sort -@ ${split_cpus} -O bam > "${libraryid}"_"${seqtype}".mapped.bam
samtools index "${libraryid}"_"${seqtype}".mapped.bam ${size}
"""
NextFlow SAMtools Bowtie 2 From line 1468 of master/main.nf 
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"""
samtools index $bam
extract_map_reads.py $bam ${r1} -m ${params.hostremoval_mode} $merged -of $out_fwd -t ${task.cpus} 
"""
NextFlow SAMtools From line 1537 of master/main.nf 
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"""
samtools index $bam
extract_map_reads.py $bam ${r1} -rev ${r2} -m ${params.hostremoval_mode} $merged -of $out_fwd -or $out_rev -t ${task.cpus}
"""
NextFlow SAMtools From line 1544 of master/main.nf 
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"""
samtools merge ${libraryid}_seqtypemerged.bam ${bam}
samtools index ${libraryid}_seqtypemerged.bam ${size}
"""
NextFlow SAMtools From line 1602 of master/main.nf 
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"""
samtools flagstat $bam > ${libraryid}_flagstat.stats
"""
NextFlow SAMtools From line 1628 of master/main.nf 
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"""
samtools view -h ${bam} -@ ${task.cpus} -q ${params.bam_mapping_quality_threshold} -b > ${libraryid}.filtered.bam
samtools index ${libraryid}.filtered.bam ${size}
"""
NextFlow SAMtools From line 1664 of master/main.nf 
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"""
samtools view -h ${bam} -@ ${task.cpus} -F4 -q ${params.bam_mapping_quality_threshold} -b > ${libraryid}.filtered.bam
samtools index ${libraryid}.filtered.bam ${size}
"""
NextFlow SAMtools From line 1669 of master/main.nf 
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"""
samtools view -h ${bam} -@ ${task.cpus} -f4 -b > ${libraryid}.unmapped.bam
samtools view -h ${bam} -@ ${task.cpus} -F4 -q ${params.bam_mapping_quality_threshold} -b > ${libraryid}.filtered.bam
samtools index ${libraryid}.filtered.bam ${size}
"""
NextFlow SAMtools From line 1674 of master/main.nf 
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"""
samtools view -h ${bam} -@ ${task.cpus} -f4 -b > ${libraryid}.unmapped.bam
samtools view -h ${bam} -@ ${task.cpus} -F4 -q ${params.bam_mapping_quality_threshold} -b > ${libraryid}.filtered.bam
samtools index ${libraryid}.filtered.bam ${size}

## FASTQ
samtools fastq -tN ${libraryid}.unmapped.bam | pigz -p ${task.cpus - 1} > ${libraryid}.unmapped.fastq.gz
rm ${libraryid}.unmapped.bam
"""
NextFlow SAMtools From line 1680 of master/main.nf 
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"""
samtools view -h ${bam} -@ ${task.cpus} -f4 -b > ${libraryid}.unmapped.bam
samtools view -h ${bam} -@ ${task.cpus} -F4 -q ${params.bam_mapping_quality_threshold} -b > ${libraryid}.filtered.bam
samtools index ${libraryid}.filtered.bam ${size}

## FASTQ
samtools fastq -tN ${libraryid}.unmapped.bam | pigz -p ${task.cpus -1} > ${libraryid}.unmapped.fastq.gz
"""
NextFlow SAMtools From line 1690 of master/main.nf 
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"""
samtools view -h ${bam} -@ ${task.cpus} -q ${params.bam_mapping_quality_threshold} -b > tmp_mapped.bam
filter_bam_fragment_length.py -a -l ${params.bam_filter_minreadlength} -o ${libraryid} tmp_mapped.bam
samtools index ${libraryid}.filtered.bam ${size}
"""
NextFlow SAMtools From line 1700 of master/main.nf 
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"""
samtools view -h ${bam} -@ ${task.cpus} -F4 -q ${params.bam_mapping_quality_threshold} -b > tmp_mapped.bam
filter_bam_fragment_length.py -a -l ${params.bam_filter_minreadlength} -o ${libraryid} tmp_mapped.bam
samtools index ${libraryid}.filtered.bam ${size}
"""
NextFlow SAMtools From line 1706 of master/main.nf 
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"""
samtools view -h ${bam} -@ ${task.cpus} -f4 -b > ${libraryid}.unmapped.bam
samtools view -h ${bam} -@ ${task.cpus} -F4 -q ${params.bam_mapping_quality_threshold} -b > tmp_mapped.bam
filter_bam_fragment_length.py -a -l ${params.bam_filter_minreadlength} -o ${libraryid} tmp_mapped.bam
samtools index ${libraryid}.filtered.bam ${size}
"""
NextFlow SAMtools From line 1712 of master/main.nf 
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"""
samtools view -h ${bam} -@ ${task.cpus} -f4 -b > ${libraryid}.unmapped.bam
samtools view -h ${bam} -@ ${task.cpus} -F4 -q ${params.bam_mapping_quality_threshold} -b > tmp_mapped.bam
filter_bam_fragment_length.py -a -l ${params.bam_filter_minreadlength} -o ${libraryid} tmp_mapped.bam
samtools index ${libraryid}.filtered.bam ${size}

## FASTQ
samtools fastq -tN ${libraryid}.unmapped.bam | pigz -p ${task.cpus - 1} > ${libraryid}.unmapped.fastq.gz
rm ${libraryid}.unmapped.bam
"""
NextFlow SAMtools From line 1719 of master/main.nf 
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"""
samtools view -h ${bam} -@ ${task.cpus} -f4 -b > ${libraryid}.unmapped.bam
samtools view -h ${bam} -@ ${task.cpus} -F4 -q ${params.bam_mapping_quality_threshold} -b > tmp_mapped.bam
filter_bam_fragment_length.py -a -l ${params.bam_filter_minreadlength} -o ${libraryid} tmp_mapped.bam
samtools index ${libraryid}.filtered.bam ${size}

## FASTQ
samtools fastq -tN ${libraryid}.unmapped.bam | pigz -p ${task.cpus} > ${libraryid}.unmapped.fastq.gz
"""
NextFlow SAMtools From line 1730 of master/main.nf 
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"""
samtools flagstat $bam > ${libraryid}_postfilterflagstat.stats
"""
NextFlow SAMtools From line 1771 of master/main.nf 
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"""
endorS.py -o json -n ${libraryid} ${stats} ${poststats}
"""
NextFlow JSON From line 1815 of master/main.nf 
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"""
endorS.py -o json -n ${libraryid} ${stats}
"""
NextFlow JSON From line 1819 of master/main.nf 
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"""
mv ${bam} ${libraryid}.bam
dedup -Xmx${task.memory.toGiga()}g -i ${libraryid}.bam $treat_merged -o . -u 
mv *.log dedup.log
samtools sort -@ ${task.cpus} "${libraryid}"_rmdup.bam -o "${libraryid}"_rmdup.bam
samtools index "${libraryid}"_rmdup.bam ${size}
"""
NextFlow SAMtools From line 1850 of master/main.nf 
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"""
dedup -Xmx${task.memory.toGiga()}g -i ${libraryid}.bam $treat_merged -o . -u 
mv *.log dedup.log
samtools sort -@ ${task.cpus} "${libraryid}"_rmdup.bam -o "${libraryid}"_rmdup.bam
samtools index "${libraryid}"_rmdup.bam ${size}
"""
NextFlow SAMtools From line 1858 of master/main.nf 
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"""
mv ${bam} ${libraryid}.bam
picard -Xmx${task.memory.toMega()}M MarkDuplicates INPUT=${libraryid}.bam OUTPUT=${libraryid}_rmdup.bam REMOVE_DUPLICATES=TRUE AS=TRUE METRICS_FILE="${libraryid}_rmdup.metrics" VALIDATION_STRINGENCY=SILENT
samtools index ${libraryid}_rmdup.bam ${size}
"""
NextFlow SAMtools Picard From line 1888 of master/main.nf 
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"""
picard -Xmx${task.memory.toMega()}M MarkDuplicates INPUT=${libraryid}.bam OUTPUT=${libraryid}_rmdup.bam REMOVE_DUPLICATES=TRUE AS=TRUE METRICS_FILE="${libraryid}_rmdup.metrics" VALIDATION_STRINGENCY=SILENT
samtools index ${libraryid}_rmdup.bam ${size}
"""
NextFlow SAMtools Picard From line 1894 of master/main.nf 
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"""
samtools merge ${samplename}_udg${udg}_libmerged_rmdup.bam ${bam}
samtools index ${samplename}_udg${udg}_libmerged_rmdup.bam ${size}
"""
NextFlow SAMtools From line 1972 of master/main.nf 
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"""
preseq c_curve -s ${params.preseq_step_size} -o ${input.baseName}.preseq -H ${input}
"""
NextFlow preseq From line 2022 of master/main.nf 
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"""
preseq c_curve -s ${params.preseq_step_size} -o ${input.baseName}.preseq -B ${input} ${pe_mode}
"""
NextFlow preseq From line 2026 of master/main.nf 
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"""
preseq c_curve -s ${params.preseq_step_size} -o ${input.baseName}.preseq -B ${input} ${pe_mode}
"""
NextFlow preseq From line 2030 of master/main.nf 
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"""
preseq lc_extrap -s ${params.preseq_step_size} -o ${input.baseName}.preseq -H ${input} -n ${params.preseq_bootstrap} -e ${params.preseq_maxextrap} -cval ${params.preseq_cval} -x ${params.preseq_terms}
"""
NextFlow preseq From line 2034 of master/main.nf 
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"""
preseq lc_extrap -s ${params.preseq_step_size} -o ${input.baseName}.preseq -B ${input} ${pe_mode} -n ${params.preseq_bootstrap} -e ${params.preseq_maxextrap} -cval ${params.preseq_cval} -x ${params.preseq_terms}
"""
NextFlow preseq From line 2038 of master/main.nf 
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"""
preseq lc_extrap -s ${params.preseq_step_size} -o ${input.baseName}.preseq -B ${input} ${pe_mode} -n ${params.preseq_bootstrap} -e ${params.preseq_maxextrap} -cval ${params.preseq_cval} -x ${params.preseq_terms}
"""
NextFlow preseq From line 2042 of master/main.nf 
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"""
## Create genome file from bam header
samtools view -H ${bam} | grep '@SQ' | sed 's#@SQ\tSN:\\|LN:##g' > genome.txt

##  Run bedtools
bedtools coverage -nonamecheck -g genome.txt -sorted -a ${anno_file} -b ${bam} | pigz -p ${task.cpus - 1} > "${bam.baseName}".breadth.gz
bedtools coverage -nonamecheck -g genome.txt -sorted -a ${anno_file} -b ${bam} -mean | pigz -p ${task.cpus - 1} > "${bam.baseName}".depth.gz
"""
NextFlow SAMtools BEDTools From line 2066 of master/main.nf 
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"""
damageprofiler -Xmx${task.memory.toGiga()}g -i $bam -r $fasta -l ${params.damageprofiler_length} -t ${params.damageprofiler_threshold} -o . -yaxis_damageplot ${params.damageprofiler_yaxis}
"""
NextFlow From line 2104 of master/main.nf 
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"""
mapDamage -i ${bam} -r ${fasta} --rescale --rescale-out="${base}_rescaled.bam" --seq-length=${params.rescale_seqlength} ${rescale_length_5p} ${rescale_length_3p} ${singlestranded}
samtools index ${base}_rescaled.bam ${size}
"""
NextFlow SAMtools MapDamage From line 2134 of master/main.nf 
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"""
bedtools maskfasta -fi ${fasta} -bed ${bedfile} -fo ${fasta.baseName}_masked.fa
"""
NextFlow BEDTools From line 2159 of master/main.nf 
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"""
#Run Filtering step 
samtools calmd ${bam} ${fasta} | pmdtools --threshold ${params.pmdtools_threshold} ${treatment} --header | samtools view -Sb - > "${libraryid}".pmd.bam

#Run Calc Range step
## To allow early shut off of pipe: https://github.com/nextflow-io/nextflow/issues/1564
trap 'if [[ \$? == 141 ]]; then echo "Shutting samtools early due to -n parameter" && samtools index ${libraryid}.pmd.bam ${size}; exit 0; fi' EXIT
samtools calmd ${bam} ${fasta} | pmdtools --deamination ${platypus} --range ${params.pmdtools_range} ${treatment} -n ${params.pmdtools_max_reads} > "${libraryid}".cpg.range."${params.pmdtools_range}".txt

samtools index ${libraryid}.pmd.bam ${size}
"""
NextFlow SAMtools pmdtools From line 2191 of master/main.nf 
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"""
bam trimBam $bam tmp.bam -L ${left_clipping} -R ${right_clipping} ${softclip}
samtools sort -@ ${task.cpus} tmp.bam -o ${libraryid}.trimmed.bam 
samtools index ${libraryid}.trimmed.bam ${size}
"""
NextFlow SAMtools From line 2246 of master/main.nf 
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"""
samtools merge ${samplename}_libmerged_add.bam ${bam}
samtools index ${samplename}_libmerged_add.bam ${size}
"""
NextFlow SAMtools From line 2296 of master/main.nf 
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"""
qualimap bamqc -bam $bam -nt ${task.cpus} -outdir . -outformat "HTML" ${snpcap} --java-mem-size=${task.memory.toGiga()}G
"""
NextFlow QualiMap From line 2326 of master/main.nf 
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"""
picard -Xmx${task.memory.toGiga()}g AddOrReplaceReadGroups I=${bam} O=${samplename}_rg.bam RGID=1 RGLB="${samplename}_rg" RGPL=illumina RGPU=4410 RGSM="${samplename}_rg" VALIDATION_STRINGENCY=LENIENT
samtools index ${samplename}_rg.bam ${size}
"""
NextFlow SAMtools Picard From line 2392 of master/main.nf 
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"""
samtools index -b ${bam}
gatk3 -Xmx${task.memory.toGiga()}g -T RealignerTargetCreator -R ${fasta} -I ${bam} -nt ${task.cpus} -o ${samplename}.intervals ${defaultbasequalities}
gatk3 -Xmx${task.memory.toGiga()}g -T IndelRealigner -R ${fasta} -I ${bam} -targetIntervals ${samplename}.intervals -o ${samplename}.realign.bam ${defaultbasequalities}
gatk3 -Xmx${task.memory.toGiga()}g -T UnifiedGenotyper -R ${fasta} -I ${samplename}.realign.bam -o ${samplename}.unifiedgenotyper.vcf -nt ${task.cpus} --genotype_likelihoods_model ${params.gatk_ug_genotype_model} -stand_call_conf ${params.gatk_call_conf} --sample_ploidy ${params.gatk_ploidy} -dcov ${params.gatk_downsample} --output_mode ${params.gatk_ug_out_mode} ${defaultbasequalities}

$keep_realign

bgzip -@ ${task.cpus} ${samplename}.unifiedgenotyper.vcf
"""
NextFlow SAMtools gatk gatk_realigner_target_creator From line 2429 of master/main.nf 
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"""
samtools index ${bam}
gatk3 -Xmx${task.memory.toGiga()}g -T RealignerTargetCreator -R ${fasta} -I ${bam} -nt ${task.cpus} -o ${samplename}.intervals ${defaultbasequalities}
gatk3 -Xmx${task.memory.toGiga()}g -T IndelRealigner -R ${fasta} -I ${bam} -targetIntervals ${samplenane}.intervals -o ${samplename}.realign.bam ${defaultbasequalities}
gatk3 -Xmx${task.memory.toGiga()}g -T UnifiedGenotyper -R ${fasta} -I ${samplename}.realign.bam -o ${samplename}.unifiedgenotyper.vcf -nt ${task.cpus} --dbsnp ${params.gatk_dbsnp} --genotype_likelihoods_model ${params.gatk_ug_genotype_model} -stand_call_conf ${params.gatk_call_conf} --sample_ploidy ${params.gatk_ploidy} -dcov ${params.gatk_downsample} --output_mode ${params.gatk_ug_out_mode} ${defaultbasequalities}

$keep_realign

bgzip -@  ${task.cpus} ${samplename}.unifiedgenotyper.vcf
"""
NextFlow SAMtools gatk gatk_realigner_target_creator From line 2440 of master/main.nf 
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"""
gatk HaplotypeCaller --java-options "-Xmx${task.memory.toGiga()}G" -R ${fasta} -I ${bam} -O ${samplename}.haplotypecaller.vcf -stand-call-conf ${params.gatk_call_conf} --sample-ploidy ${params.gatk_ploidy} --output-mode ${params.gatk_hc_out_mode} --emit-ref-confidence ${params.gatk_hc_emitrefconf}
bgzip -@ ${task.cpus} ${samplename}.haplotypecaller.vcf
"""
NextFlow gatk From line 2473 of master/main.nf 
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"""
gatk HaplotypeCaller --java-options "-Xmx${task.memory.toGiga()}G" -R ${fasta} -I ${bam} -O ${samplename}.haplotypecaller.vcf --dbsnp ${params.gatk_dbsnp} -stand-call-conf ${params.gatk_call_conf} --sample_ploidy ${params.gatk_ploidy} --output_mode ${params.gatk_hc_out_mode} --emit-ref-confidence ${params.gatk_hc_emitrefconf}
bgzip -@  ${task.cpus} ${samplename}.haplotypecaller.vcf
"""
NextFlow gatk From line 2479 of master/main.nf 
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"""
freebayes -f ${fasta} -p ${params.freebayes_p} -C ${params.freebayes_C} ${skip_coverage} ${bam} > ${samplename}.freebayes.vcf
bgzip -@  ${task.cpus} ${samplename}.freebayes.vcf
"""
NextFlow FreeBayes From line 2506 of master/main.nf 
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"""
samtools mpileup -B --ignore-RG -q ${map_q} -Q ${base_q} ${use_bed} -f ${fasta} ${bam_list} | pileupCaller ${caller} ${ssmode} ${transitions_mode} --sampleNames ${sample_names} ${use_snp} -e pileupcaller.${strandedness}
"""
NextFlow SAMtools From line 2587 of master/main.nf 
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"""
eigenstrat_snp_coverage -i pileupcaller.${strandedness} >${strandedness}_eigenstrat_coverage.txt -j ${strandedness}_eigenstrat_coverage_mqc.json
"""
NextFlow From line 2610 of master/main.nf 
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"""
eigenstrat_snp_coverage -i pileupcaller.${strandedness} >${strandedness}_eigenstrat_coverage.txt
parse_snp_cov.py ${strandedness}_eigenstrat_coverage.txt
"""
NextFlow From line 2614 of master/main.nf 
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"""
echo ${bam} > bam.filelist
mkdir angsd
angsd -bam bam.filelist -nThreads ${task.cpus} -GL ${angsd_glmodel} -doGlF ${angsd_glformat} ${angsd_majorminor} ${angsd_fasta} -out ${samplename}.angsd
"""
NextFlow angsd From line 2662 of master/main.nf 
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"""
bcftools stats *.vcf.gz -F ${fasta} > ${samplename}.vcf.stats
"""
NextFlow BCFtools From line 2689 of master/main.nf 
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"""
pigz -d -f -p ${task.cpus} ${vcf}
vcf2genome -Xmx${task.memory.toGiga()}g -draft ${out} -draftname "${fasta_head}" -in ${vcf.baseName} -minc ${params.vcf2genome_minc} -minfreq ${params.vcf2genome_minfreq} -minq ${params.vcf2genome_minq} -ref ${fasta} -refMod ${out}_refmod.fasta -uncertain ${out}_uncertainty.fasta
pigz -f -p ${task.cpus} ${out}*
bgzip -@ ${task.cpus} *.vcf
"""
NextFlow vcf2genome From line 2718 of master/main.nf 
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"""
pigz -d -f -p ${task.cpus} ${vcf}
multivcfanalyzer -Xmx${task.memory.toGiga()}g ${params.snp_eff_results} ${fasta} ${params.reference_gff_annotations} . ${write_freqs} ${params.min_genotype_quality} ${params.min_base_coverage} ${params.min_allele_freq_hom} ${params.min_allele_freq_het} ${params.reference_gff_exclude} *.vcf
pigz -p ${task.cpus} *.tsv *.txt snpAlignment.fasta snpAlignmentIncludingRefGenome.fasta fullAlignment.fasta
bgzip -@ ${task.cpus} *.vcf
"""
NextFlow From line 2761 of master/main.nf 
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"""
mtnucratio -Xmx${task.memory.toGiga()}g ${bam} "${params.mtnucratio_header}"
"""
NextFlow mtnucratio From line 2791 of master/main.nf 
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"""
mv ${bam} ${bam.baseName}_${strandedness}strand.bam
"""
NextFlow From line 2812 of master/main.nf 
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"""
ls *.bam >> bamlist.txt
samtools depth -aa -q30 -Q30 $filter -f bamlist.txt | sexdeterrmine -f bamlist.txt > SexDet.txt
"""
NextFlow SAMtools From line 2836 of master/main.nf 
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"""
samtools index ${input}
angsd -i ${input} -r ${params.contamination_chrom_name}:5000000-154900000 -doCounts 1 -iCounts 1 -minMapQ 30 -minQ 30 -out ${libraryid}.doCounts
contamination -a ${libraryid}.doCounts.icnts.gz -h ${projectDir}/assets/angsd_resources/HapMapChrX.gz 2> ${libraryid}.X.contamination.out
"""
NextFlow SAMtools angsd From line 2859 of master/main.nf 
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"""
print_x_contamination.py ${Contam.join(' ')}
"""
NextFlow From line 2882 of master/main.nf 
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"""
bbduk.sh -Xmx${task.memory.toGiga()}g in=${fastq} threads=${task.cpus} entropymask=f entropy=${params.metagenomic_complexity_entropy} out=${fastq}_lowcomplexityremoved.fq.gz 2> ${fastq}_bbduk.stats
"""
NextFlow BBMap From line 2910 of master/main.nf 
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"""
malt-run \
-J-Xmx${task.memory.toGiga()}g \
-t ${task.cpus} \
-v \
-o . \
-d ${db} \
${sam_out} \
-id ${params.percent_identity} \
-m ${params.malt_mode} \
-at ${params.malt_alignment_mode} \
-top ${params.malt_top_percent} \
${min_supp} \
-mq ${params.malt_max_queries} \
--memoryMode ${params.malt_memory_mode} \
-i ${fastqs.join(' ')} |&tee malt.log
"""
NextFlow Malt From line 2969 of master/main.nf 
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"""
MaltExtract \
-Xmx${task.memory.toGiga()}g \
-t ${taxon_list} \
-i ${rma6.join(' ')} \
-o results/ \
-r ${ncbifiles} \
-p ${task.cpus} \
-f ${params.maltextract_filter} \
-a ${params.maltextract_toppercent} \
--minPI ${params.maltextract_percentidentity} \
${destack} \
${downsam} \
${dupremo} \
${matches} \
${megsum} \
${topaln} \
${ss}

postprocessing.AMPS.r -r results/ -m ${params.maltextract_filter} -t ${task.cpus} -n ${taxon_list} -j
"""
NextFlow AMPS From line 3015 of master/main.nf 
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"""
tar xvzf $ckdb
mkdir -p $dbname
mv *.k2d $dbname || echo "nothing to do"
"""
NextFlow From line 3052 of master/main.nf 
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"""
kraken2 --db ${krakendb} --threads ${task.cpus} --output $out --report-minimizer-data --report $kreport $fastq
cut -f1-3,6-8 $kreport > $kreport_old
"""
NextFlow kraken2 From line 3087 of master/main.nf 
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"""
kraken_parse.py -c ${params.metagenomic_min_support_reads} -or $read_out -ok $kmer_out $kraken_r
"""
NextFlow From line 3106 of master/main.nf 
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"""
merge_kraken_res.py -or $read_out -ok $kmer_out
"""
NextFlow From line 3123 of master/main.nf 
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"""
markdown_to_html.py $output_docs -o results_description.html
"""
NextFlow From line 3146 of master/main.nf 
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"""
echo $workflow.manifest.version &> v_pipeline.txt
echo $workflow.nextflow.version &> v_nextflow.txt

fastqc -t ${task.cpus} --version &> v_fastqc.txt 2>&1 || true
AdapterRemoval --version  &> v_adapterremoval.txt 2>&1 || true
fastp --version &> v_fastp.txt 2>&1 || true
bwa &> v_bwa.txt 2>&1 || true
circulargenerator -Xmx${task.memory.toGiga()}g --help | head -n 1 &> v_circulargenerator.txt 2>&1 || true
samtools --version &> v_samtools.txt 2>&1 || true
dedup -Xmx${task.memory.toGiga()}g -v &> v_dedup.txt 2>&1 || true
## bioconda recipe of picard is incorrectly set up and extra warning made with stderr, this ugly command ensures only version exported
( exec 7>&1; picard -Xmx${task.memory.toMega()}M MarkDuplicates --version 2>&1 >&7 | grep -v '/' >&2 ) 2> v_markduplicates.txt || true
qualimap --version --java-mem-size=${task.memory.toGiga()}G &> v_qualimap.txt 2>&1 || true
preseq &> v_preseq.txt 2>&1 || true
gatk --java-options "-Xmx${task.memory.toGiga()}G" --version 2>&1 | grep '(GATK)' > v_gatk.txt 2>&1 || true
gatk3 -Xmx${task.memory.toGiga()}g  --version 2>&1 | head -n 1 > v_gatk3.txt 2>&1 || true
freebayes --version &> v_freebayes.txt 2>&1 || true
bedtools --version &> v_bedtools.txt 2>&1 || true
damageprofiler -Xmx${task.memory.toGiga()}g --version &> v_damageprofiler.txt 2>&1 || true
bam --version &> v_bamutil.txt 2>&1 || true
pmdtools --version &> v_pmdtools.txt 2>&1 || true
angsd -h |& head -n 1 | cut -d ' ' -f3-4 &> v_angsd.txt 2>&1 || true 
multivcfanalyzer -Xmx${task.memory.toGiga()}g --help | head -n 1 &> v_multivcfanalyzer.txt 2>&1 || true
malt-run -J-Xmx${task.memory.toGiga()}g --help |& tail -n 3 | head -n 1 | cut -f 2 -d'(' | cut -f 1 -d ',' &> v_malt.txt 2>&1 || true
MaltExtract -Xmx${task.memory.toGiga()}g --help | head -n 2 | tail -n 1 &> v_maltextract.txt 2>&1 || true
multiqc --version &> v_multiqc.txt 2>&1 || true
vcf2genome -Xmx${task.memory.toGiga()}g -h |& head -n 1 &> v_vcf2genome.txt || true
mtnucratio -Xmx${task.memory.toGiga()}g --help &> v_mtnucratiocalculator.txt || true
sexdeterrmine --version &> v_sexdeterrmine.txt || true
kraken2 --version | head -n 1 &> v_kraken.txt || true
endorS.py --version &> v_endorSpy.txt || true
pileupCaller --version &> v_sequencetools.txt 2>&1 || true
bowtie2 --version | grep -a 'bowtie2-.* -fdebug' > v_bowtie2.txt || true
eigenstrat_snp_coverage --version | cut -d ' ' -f2 >v_eigenstrat_snp_coverage.txt || true
mapDamage --version > v_mapdamage.txt || true
bbversion.sh > v_bbduk.txt || true
bcftools --version | grep 'bcftools' | cut -d ' ' -f 2 > v_bcftools.txt || true
scrape_software_versions.py &> software_versions_mqc.yaml
"""
NextFlow SAMtools gatk FastQC BCFtools MultiQC BEDTools BWA Picard Bowtie 2 fastp kraken2 BBMap FreeBayes QualiMap preseq AdapterRemoval pmdtools angsd Malt MapDamage mtnucratio vcf2genome From line 3168 of master/main.nf 
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"""
multiqc -f $rtitle $rfilename $multiqc_config $custom_config_file .
"""
NextFlow MultiQC From line 3263 of master/main.nf
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Created: 1yr ago
Updated: 1yr ago
Maitainers: public
URL: https://nf-co.re/eager
Name: eager
Version: 2.4.7
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