This repository contains a nextflow workflow for analysing variation in human genomic data. Specifically this workflow can perform the following:

• basecalling of FAST5 (or POD5) sequencing data
• diploid variant calling
• structural variant calling
• aggregation of modified base counts
• copy number variant calling
• short tandem repeat (STR) expansion genotyping

The wf-human-variation workflow consolidates the small variant calling from the previous wf-human-snp, structural variant calling from wf-human-sv, CNV calling from wf-cnv (all of which are now deprecated), as well as performing STR expansion genotyping. This pipeline performs the steps of the four pipelines simultaneously and the results are generated and output in the same way as they would have been had the pipelines been run separately.

This workflow uses Clair3 for calling small variants from long reads. Clair3 makes the best of two methods: pileup (for fast calling of variant candidates in high confidence regions), and full-alignment (to improve precision of calls of more complex candidates).

This workflow uses sniffles2 for calling structural variants.

This workflow uses modbam2bed to aggregate modified base counts into a bedMethyl file.

This workflow uses Dorado for basecalling pod5 or fast5 signal data.

This workflow uses QDNAseq for calling copy number variants.

This workflow uses a fork of Straglr for genotyping short tandem repeat expansions.

The workflow uses Nextflow to manage compute and software resources, as such Nextflow will need to be installed before attempting to run the workflow.

The workflow can currently be run using either Docker or Singularity to provide isolation of the required software. Both methods are automated out-of-the-box provided either Docker or Singularity is installed.

It is not required to clone or download the git repository in order to run the workflow. For more information on running EPI2ME Labs workflows visit our website.

Workflow options

To obtain the workflow, having installed nextflow, users can run:

nextflow run epi2me-labs/wf-human-variation --help

to see the options for the workflow.

Download demonstration data

A small test dataset is provided for the purposes of testing the workflow software, it can be downloaded using:

wget -O demo_data.tar.gz \
    https://ont-exd-int-s3-euwst1-epi2me-labs.s3.amazonaws.com/wf-human-variation/demo_data.tar.gz
tar -xzvf demo_data.tar.gz

The basecalling, SNP, SV, 5mC aggregation, and CNV workflows are all independent and can be run in isolation or together using options to activate them. The STR workflow can also be run independently but will trigger the SNP workflow to run first, as a phased VCF is required to haplotag the input BAM file in order to successfully perform STR genotyping.

The SNP and SV workflows can be run with the demonstration data using:

OUTPUT=output
nextflow run epi2me-labs/wf-human-variation \
    -w ${OUTPUT}/workspace \
    -profile standard \
    --snp --sv \
    --bam demo_data/demo.bam \
    --bed demo_data/demo.bed \
    --ref demo_data/demo.fasta \
    --basecaller_cfg '[email protected]'  \
    --sample_name MY_SAMPLE \
    --out_dir ${OUTPUT}

Each subworkflow is enabled with a command line option:

• Basecalling: --fast5_dir <input_dir>
• SNP calling: --snp
• SV calling: --sv
• Methylation aggregation: --methyl
• CNV calling: --cnv
• STR genotyping: --str

Subworkflows where the relevant option is omitted will not be run.

Some subworkflows have additional required options:

• The SV workflow takes an optional --tr_bed option to specify tandem repeats in the reference sequence --- see the sniffles documentation for more information.

• The STR workflow takes a required --sex option which is male or female. If --sex is not specified, the workflow will default to female. Please be aware that incorrect sex assignment will result in the wrong number of calls for all repeats on chrX.

To enable the 5mC aggregation step use the --methyl option. For this step to produce meaningful output the input BAM file must have been produced by a basecaller capable of emitting the 5mC calls.

This brings us to activating the basecalling workflow. To run all the above including basecalling:

OUTPUT=output
nextflow run epi2me-labs/wf-human-variation \
    -w ${OUTPUT}/workspace \
    -profile standard \
    --snp --sv --methyl \
    --fast5_dir path/to/fast5/dir \
    --basecaller_cfg '[email protected]'  \
    --remora_cfg '[email protected]_5mCG@v2' \
    --bed path/to.bed \
    --ref path/to.fasta \
    --out_dir ${OUTPUT}

Workflow outputs

The primary outputs of the workflow include:

• a gzipped VCF file containing SNPs found in the dataset (--snp)
• a gzipped VCF file containing the SVs called from the dataset (--sv)
• a gzipped bedMethyl file aggregating modified CpG base counts (--methyl)
• a VCF of CNV calls, QDNAseq-generated plots, and BED files of both raw read counts per bin and corrected, normalised, and smoothed read counts per bin (--cnv)
• a gzipped VCF file containing STRs found in the dataset, TSV Straglr output containing reads spanning STRs, and a haplotagged BAM (--str)
• an HTML report detailing the primary findings of the workflow, for SNP, SV, CNV calling, and STR genotyping.
• if basecalling and alignment was conducted, the workflow will output two sorted, indexed CRAMs of basecalls aligned to the provided references, with reads separated by their quality score:
  • <sample_name>.pass.cram contains reads with qscore >= threshold (only pass reads are used to make downstream variant cals)
  • <sample_name>.fail.cram contains reads with qscore < threshold
• if unaligned reads were provided, the workflow will output a CRAM file containing the alignments used to make the downstream variant calls

The secondary outputs of the workflow include:

• {sample_name}.mapula.csv and {sample_name}.mapula.json provide basic alignment metrics (primary and secondary counts, read N50, median accuracy)
• mosdepth outputs include:
  • {sample_name}.mosdepth.global.dist.txt: a cumulative distribution indicating the proportion of total bases for each and all reference sequences more info
  • {sample_name}.regions.bed.gz: reports the mean coverage for each region in the provided BED file
  • {sample_name}.thresholds.bed.gz: reports the number of bases in each region that are covered at or above each threshold value (1, 10, 20, 30X) more info
• {sample_name}.readstats.tsv.gz: a gzipped TSV summarising per-alignment statistics produced by bamstats

Workflow tips

• Users familiar with wf-human-snp and wf-human-sv are recommended to familiarise themselves with any parameter changes by using --help, in particular:
  • All arms of the variation calling workflow use --ref (not --reference) and --bed (not --target_bedfile)
• Specifying a suitable tandem repeat BED for your reference with --tr_bed can improve the accuracy of SV calling.
• Aggregation of methylation calls with --methyl requires data to be basecalled with a model that includes base modifications, providing the MM and ML BAM tags
• Refer to the Dorado documentation for a list of available basecalling models
• Take care to retain the input reference when basecalling or alignment has been performed as CRAM files cannot be read without the corresponding reference!
• Refer to our blogpost and CNV workflow documentation for more information on running the copy number calling subworkflow.
• The STR workflow performs genotyping of specific repeats, which can be found here, and is only compatible with genome build 38.

• Nextflow
• Docker
• Singularity