Pipeline to produce consensus reads using unique molecular indexes/barcodes (UMIs)

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Introduction

nf-core/fastquorum is a bioinformatics best-practice analysis pipeline to produce consensus reads using unique molecular indexes/barcodes (UMIs). The pipeline implements the [fgbio Best Practices FASTQ to Consensus Pipeline][fgbio-best-practices-link]. fastquorum can produce consensus reads from single or multi UMI reads, and even [Duplex Sequencing][duplex-seq-link] reads.

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. Fastq to BAM, extracting UMIs ( fgbio FastqToBam )

  3. Align ( bwa mem ), reformat ( fgbio ZipperBam ), and template-coordinate sort ( samtools sort )

  4. Group reads by UMI ( fgbio GroupReadsByUmi )

  5. Call consensus reads

    1. For [Duplex-Sequencing][duplex-seq-link] data

      1. Call duplex consensus reads ( fgbio CallDuplexConsensusReads )

      2. Collect duplex sequencing specific metrics ( fgbio CollectDuplexSeqMetrics )

    2. For non-Duplex-Sequencing data:

      1. Call molecular consensus reads ( fgbio CallMolecularConsensusReads )

  6. Align ( bwa mem )

  7. Filter consensus reads ( fgbio FilterConsensusReads )

  8. Present QC for raw reads ( MultiQC )

Quick Start

  1. Install Nextflow ( >=21.10.3 )

  2. Install any of Docker , Singularity (you can follow this tutorial ), Podman , Shifter or Charliecloud for full pipeline reproducibility (you can use Conda both to install Nextflow itself and also to manage software within pipelines. Please only use it within pipelines as a last resort; see docs ) .

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

    nextflow run nf-core/fastquorum -profile test,YOURPROFILE --outdir <OUTDIR>

    Note that some form of configuration will be needed so that Nextflow knows how to fetch the required software. This is usually done in the form of a config profile ( YOURPROFILE in the example command above). You can chain multiple config profiles in a comma-separated string.

    • The pipeline comes with config profiles called docker , singularity , podman , shifter , charliecloud and conda which instruct the pipeline to use the named tool for software management. For example, -profile test,docker .

    • Please check nf-core/configs to see if a custom config file to run nf-core pipelines already exists for your Institute. If so, you can simply use -profile <institute> in your command. This will enable either docker or singularity and set the appropriate execution settings for your local compute environment.

    • If you are using singularity , please use the nf-core download command to download images first, before running the pipeline. Setting the NXF_SINGULARITY_CACHEDIR or singularity.cacheDir Nextflow options enables you to store and re-use the images from a central location for future pipeline runs.

    • If you are using conda , it is highly recommended to use the NXF_CONDA_CACHEDIR or conda.cacheDir settings to store the environments in a central location for future pipeline runs.

  4. Start running your own analysis! 

    nextflow run nf-core/fastquorum --input samplesheet.csv --outdir <OUTDIR> --genome GRCh37 -profile <docker/singularity/podman/shifter/charliecloud/conda/institute>

Documentation

The nf-core/fastquorum pipeline comes with documentation about the pipeline usage , parameters and output . m See also:

  1. The [fgbio Best Practise FASTQ -> Consensus Pipeline][fgbio-best-practices-link]

  2. Read structures as required in the input sample sheet.

Credits

nf-core/fastquorum was originally written by Nils Homer.

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

Acknowledgements

Fulcrum Genomics

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 #fastquorum 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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"""
# The real path to the FASTA
FASTA=`find -L ./ -name "*.amb" | sed 's/.amb//'`

samtools fastq ${samtools_fastq_args} ${unmapped_bam} \\
    | bwa mem ${bwa_args} -t $task.cpus -p -K 150000000 -Y \$FASTA - \\
    | fgbio -Xmx${fgbio_mem_gb}g \\
        --compression ${fgbio_zipper_bams_compression} \\
        --async-io=true \\
        ZipperBams \\
        --unmapped ${unmapped_bam} \\
        --ref \$FASTA \\
        --output ${fgbio_zipper_bams_output} \\
        --tags-to-reverse Consensus \\
        --tags-to-revcomp Consensus \\
        ${fgbio_args} \\
        ${extra_command};

cat <<-END_VERSIONS > versions.yml
"${task.process}":
    bwa: \$(echo \$(bwa 2>&1) | sed 's/^.*Version: //; s/Contact:.*\$//')
    fgbio: \$( echo \$(fgbio --version 2>&1 | tr -d '[:cntrl:]' ) | sed -e 's/^.*Version: //;s/\\[.*\$//')
    samtools: \$(echo \$(samtools --version 2>&1) | sed 's/^.*samtools //; s/Using.*\$//')
END_VERSIONS
"""
NextFlow SAMtools BWA fgbio From line 53 of align_bam/main.nf 
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"""
fgbio \\
    -Xmx${mem_gb}g \\
    --tmp-dir=. \\
    --async-io=true \\
    --compression=1 \\
    CallDuplexConsensusReads \\
    --input $grouped_bam \\
    --output ${prefix}.cons.unmapped.bam \\
    --min-reads ${min_reads} \\
    --min-input-base-quality ${min_baseq} \\
    --threads ${task.cpus} \\
    $args;

cat <<-END_VERSIONS > versions.yml
"${task.process}":
    fgbio: \$( echo \$(fgbio --version 2>&1 | tr -d '[:cntrl:]' ) | sed -e 's/^.*Version: //;s/\\[.*\$//')
END_VERSIONS
"""
NextFlow fgbio From line 29 of callduplexconsensusreads/main.nf 
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"""
fgbio \\
    -Xmx${mem_gb}g \\
    --tmp-dir=. \\
    --async-io=true \\
    --compression=1 \\
    CallMolecularConsensusReads \\
    --input $grouped_bam \\
    --output ${prefix}.cons.unmapped.bam \\
    --min-reads ${min_reads} \\
    --min-input-base-quality ${min_baseq} \\
    --threads ${task.cpus} \\
    $args;

cat <<-END_VERSIONS > versions.yml
"${task.process}":
    fgbio: \$( echo \$(fgbio --version 2>&1 | tr -d '[:cntrl:]' ) | sed -e 's/^.*Version: //;s/\\[.*\$//')
END_VERSIONS
"""
NextFlow fgbio From line 29 of callmolecularconsensusreads/main.nf 
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"""
fgbio \\
    -Xmx${mem_gb}g \\
    --tmp-dir=. \\
    --async-io=true \\
    --compression=1 \\
    CollectDuplexSeqMetrics \\
    --input $grouped_bam \\
    --output ${prefix}.duplex_seq_metrics \\
    --duplex-umi-counts=true \\
    $args;

cat <<-END_VERSIONS > versions.yml
"${task.process}":
    fgbio: \$( echo \$(fgbio --version 2>&1 | tr -d '[:cntrl:]' ) | sed -e 's/^.*Version: //;s/\\[.*\$//')
END_VERSIONS
"""
NextFlow fgbio From line 27 of collectduplexseqmetrics/main.nf 
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"""

fgbio \\
    -Xmx${mem_gb}g \\
    --tmp-dir=. \\
    --async-io=true \\
    --compression=1 \\
    FastqToBam \\
    --input ${fastqs} \\
    --output "${prefix}.unmapped.bam" \\
    --read-structures ${read_structure} \\
    --sample ${meta.id} \\
    --library ${meta.id} \\
    $args

cat <<-END_VERSIONS > versions.yml
"${task.process}":
    fgbio: \$( echo \$(fgbio --version 2>&1 | tr -d '[:cntrl:]' ) | sed -e 's/^.*Version: //;s/\\[.*\$//')
END_VERSIONS
"""
NextFlow fgbio From line 30 of fastqtobam/main.nf 
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"""
fgbio \\
    -Xmx${mem_gb}g \\
    --tmp-dir=. \\
    --compression=0 \\
    FilterConsensusReads \\
    --input $grouped_bam \\
    --output /dev/stdout \\
    --ref ${fasta} \\
    --min-reads ${min_reads} \\
    --min-base-quality ${min_baseq} \\
    --max-base-error-rate ${max_base_error_rate} \\
    $fgbio_args \\
    | samtools sort \\
    --threads ${task.cpus} \\
    -o ${prefix}.cons.filtered.bam##idx##${prefix}.cons.filtered.bam.bai \\
    --write-index \\
    $samtools_args;

cat <<-END_VERSIONS > versions.yml
"${task.process}":
    fgbio: \$( echo \$(fgbio --version 2>&1 | tr -d '[:cntrl:]' ) | sed -e 's/^.*Version: //;s/\\[.*\$//')
END_VERSIONS
"""
NextFlow SAMtools fgbio From line 32 of filterconsensusreads/main.nf 
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"""
fgbio \\
    -Xmx${mem_gb}g \\
    --tmp-dir=. \\
    --async-io=true \\
    --compression=1 \\
    GroupReadsByUmi \\
    --strategy $strategy \\
    --edits $edits \\
    --input $mapped_bam \\
    --output ${prefix}.grouped.bam \\
    --family-size-histogram ${prefix}.grouped-family-sizes.txt \\
    $args

cat <<-END_VERSIONS > versions.yml
"${task.process}":
    fgbio: \$( echo \$(fgbio --version 2>&1 | tr -d '[:cntrl:]' ) | sed -e 's/^.*Version: //;s/\\[.*\$//')
END_VERSIONS
"""
NextFlow fgbio From line 30 of groupreadsbyumi/main.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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"""
[ ! -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 26 of fastqc/main.nf 
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"""
[ ! -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 36 of fastqc/main.nf 
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"""
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 23 of multiqc/main.nf
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Created: 1yr ago
Updated: 1yr ago
Maitainers: public
URL: https://nf-co.re/fastquorum
Name: fastquorum
Version: dev
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