Single-stranded DNA Sequencing (SSDS) nf-core pipeline

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Introduction

nf-core/ssds is a bioinformatics best-practice analysis pipeline for Single-Stranded DNA Sequencing (SSDS) pipeline.

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

Read QC ( FastQC )
Quality trim reads ( Trimgalore )
Align with BWA MEM ( BWA )
Sort BAM ( Picard )
Index BAM ( Samtools )
Parse ssDNA / dsDNA from SSDS BAM ( SSDS )
Make coverage tracks ( Bedtools ) ( UCSC tools )
Calculate enrichment ( SSDS )
Other QC ( Deeptools )
Generate QC report( MultiQC )

Quick Start

Install Nextflow ( >=21.10.3 )
Install any of Docker , Singularity , Podman , Shifter or Charliecloud for full pipeline reproducibility (please only use Conda as a last resort; see docs )
Download the pipeline and test it on a minimal dataset with a single command:
```
nextflow run nf-core/ssds -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.

Start running your own analysis!

nextflow run nf-core/ssds -profile <docker/singularity/podman/shifter/charliecloud/conda/institute> --input samplesheet.csv --genome mm10

Documentation

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

Credits

nf-core/ssds was originally written by Kevin Brick.

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

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 #ssds 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

"""    
calculate_SPoT.py \\
    --reads_bam \"${bamids}\" \\
    --intervals all \\
    --name \"${names}\" \\
    --iname all \\
    --g ${index} \\
    --rand \\
    --o ${meta.id}

cat <<-END_VERSIONS > versions.yml
"${task.process}":
    calculate_SSDS_SPoT: \$(calculate_SPoT.py --vers)
END_VERSIONS
"""

NextFlow From line 22 of calculatespot/main.nf

"""
grep -w \\+ ${bed} >${prefix}.FWD.bed
grep -w \\- ${bed} >${prefix}.REV.bed

nF=`cat ${prefix}.FWD.bed |wc -l`
nR=`cat ${prefix}.REV.bed |wc -l`

make_SSDS_bedgraphs.py --fwd ${prefix}.FWD.bed \
                       --rev ${prefix}.REV.bed \
                       --g ${genome_index} \
                       --name ${prefix} \
                       --win ${windows_bed}

cat <<-END_VERSIONS > versions.yml
"${task.process}":
    make_SSDS_bedgraphs: \$(echo "0.8.2")
END_VERSIONS
"""

NextFlow From line 22 of generate_ssds_coverage/main.nf

"""
multiqc -f $args .

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

NextFlow MultiQC From line 22 of multiqc_ssds/main.nf

"""
parse_SSDS_BAM.py \\
    --bam $bam \\
    --name $prefix \\
    $args

cat <<-END_VERSIONS > versions.yml
"${task.process}":
    parse_SSDS_bam: \$(parse_SSDS_BAM.py --vers)
END_VERSIONS
"""

NextFlow From line 22 of parse_ssds_bam/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 17 of local/samplesheet_check.nf

"""
bedtools \\
    makewindows \\
    ${arg_input} \\
    $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 22 of makewindows/main.nf

"""
bedtools \\
    sort \\
    -i $intervals \\
    $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 21 of sort/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 19 of index/main.nf

"""
INDEX=`find -L ./ -name "*.amb" | sed 's/.amb//'`

bwa mem \\
    $args \\
    $read_group \\
    -t $task.cpus \\
    \$INDEX \\
    $reads \\
    | samtools $samtools_command $args2 --threads $task.cpus -o ${prefix}.bam -

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

NextFlow SAMtools BWA From line 25 of mem/main.nf

"""
plotFingerprint \\
    $args \\
    $extend \\
    --bamfiles ${bams.join(' ')} \\
    --plotFile ${prefix}.plotFingerprint.pdf \\
    --outRawCounts ${prefix}.plotFingerprint.raw.txt \\
    --outQualityMetrics ${prefix}.plotFingerprint.qcmetrics.txt \\
    --numberOfProcessors $task.cpus

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

NextFlow DeepTools From line 23 of plotfingerprint/main.nf

"""
[ ! -f  ${prefix}.fastq.gz ] && ln -s $reads ${prefix}.fastq.gz
fastqc $args --threads $task.cpus ${prefix}.fastq.gz

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

NextFlow FastQC From line 23 of fastqc/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
fastqc $args --threads $task.cpus ${prefix}_1.fastq.gz ${prefix}_2.fastq.gz

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

NextFlow FastQC From line 33 of fastqc/main.nf

"""
picard \\
    -Xmx${avail_mem}g \\
    CollectMultipleMetrics \\
    $args \\
    INPUT=$bam \\
    OUTPUT=${prefix}.CollectMultipleMetrics \\
    REFERENCE_SEQUENCE=$fasta

cat <<-END_VERSIONS > versions.yml
"${task.process}":
    picard: \$(picard CollectMultipleMetrics --version 2>&1 | grep -o 'Version.*' | cut -f2- -d:)
END_VERSIONS
"""

NextFlow Picard From line 28 of collectmultiplemetrics/main.nf

"""
picard \\
    -Xmx${avail_mem}g \\
    MarkDuplicates \\
    $args \\
    I=$bam \\
    O=${prefix}.bam \\
    M=${prefix}.MarkDuplicates.metrics.txt

cat <<-END_VERSIONS > versions.yml
"${task.process}":
    picard: \$(echo \$(picard MarkDuplicates --version 2>&1) | grep -o 'Version:.*' | cut -f2- -d:)
END_VERSIONS
"""

NextFlow Picard From line 28 of markduplicates/main.nf

"""
picard \\
    SortSam \\
    -Xmx${avail_mem}g \\
    --INPUT $bam \\
    --OUTPUT ${prefix}.bam \\
    --SORT_ORDER $sort_order

cat <<-END_VERSIONS > versions.yml
"${task.process}":
    picard: \$(picard SortSam --version 2>&1 | grep -o 'Version:.*' | cut -f2- -d:)
END_VERSIONS
"""

NextFlow Picard From line 27 of sortsam/main.nf

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

NextFlow SAMtools From line 19 of idxstats/main.nf

"""
samtools index -@ ${task.cpus-1} $args $input

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

NextFlow SAMtools From line 21 of index/main.nf

"""
samtools view --threads ${task.cpus-1} ${reference} $args $input > ${prefix}.${file_type}

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 view/main.nf

"""
[ ! -f  ${prefix}.fastq.gz ] && ln -s $reads ${prefix}.fastq.gz
trim_galore \\
    $args \\
    --cores $cores \\
    --gzip \\
    $c_r1 \\
    $tpc_r1 \\
    ${prefix}.fastq.gz
cat <<-END_VERSIONS > versions.yml
"${task.process}":
    trimgalore: \$(echo \$(trim_galore --version 2>&1) | sed 's/^.*version //; s/Last.*\$//')
    cutadapt: \$(cutadapt --version)
END_VERSIONS
"""

NextFlow Trim_Galore From line 43 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 \\
    $c_r1 \\
    $c_r2 \\
    $tpc_r1 \\
    $tpc_r2 \\
    ${prefix}_1.fastq.gz \\
    ${prefix}_2.fastq.gz
cat <<-END_VERSIONS > versions.yml
"${task.process}":
    trimgalore: \$(echo \$(trim_galore --version 2>&1) | sed 's/^.*version //; s/Last.*\$//')
    cutadapt: \$(cutadapt --version)
END_VERSIONS
"""

NextFlow Trim_Galore From line 59 of trimgalore/main.nf

"""
bedGraphToBigWig \\
    $bedgraph \\
    $sizes \\
    ${prefix}.bigWig

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