wf-human-variation documentation

By EPI2ME Labs
8 min read

Human variation workflow

SNV, SV and CNV calling, modified base calling, and STR genotyping of human samples.


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

  • diploid variant calling
  • structural variant calling
  • analysis of modified base calls
  • 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.

Compute requirements

Recommended requirements:

  • CPUs = 32
  • Memory = 128GB

Minimum requirements:

  • CPUs = 12
  • Memory = 32GB

Approximate run time: Variable depending on whether it is targeted sequencing or whole genome sequencing, as well as coverage and the individual analyses requested. For instance, a 90X human sample run (options: --snp --sv --mod --str --cnv --phased --sex male) takes less than 8h with recommended resources.

ARM processor support: False

Install and run

These are instructions to install and run the workflow on command line. You can also access the workflow via the EPI2ME application.

The workflow uses nextflow to manage compute and software resources, therefore 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. This is controlled by the -profile parameter as exemplified in the example below.

It is not required to clone or download the git repository in order to run the workflow. More information on running EPI2ME workflows can be found on our website.

The following command can be used to obtain the workflow. This will pull the repository in to the assets folder of nextflow and provide a list of all parameters available for the workflow as well as an example command:

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

A demo dataset is provided for testing of the workflow. It can be downloaded using:

tar -xzvf wf-human-variation-demo.tar.gz

The workflow can be run with the demo data using:

nextflow run epi2me-labs/wf-human-variation \
--bam 'wf-human-variation-demo/demo.bam' \
--basecaller_cfg 'dna_r10.4.1_e8.2_400bps_hac_prom' \
--mod \
--ref 'wf-human-variation-demo/demo.fasta' \
--sample_name 'DEMO' \
--snp \
--sv \
-profile standard

For further information about running a workflow on the command line see our workflow quick start guide.

This workflow is designed to take input sequences that have been produced from Oxford Nanopore Technologies devices.

Find related protocols in the Nanopore community.

Input example

The --bam input parameter for this workflow accepts a path to a single BAM file, or a folder containing multiple BAM files for the sample. A sample name can be supplied with --sample.

(i) (ii)
input_reads.bam ─── input_directory
├── reads0.bam
└── reads1.bam

Input parameters

Workflow Options

Nextflow parameter nameTypeDescriptionHelpDefault
svbooleanCall for structural variants.If this option is selected, structural variant calling will be carried out using Sniffles2.False
snpbooleanCall for small variantsIf this option is selected, small variant calling will be carried out using Clair3.False
cnvbooleanCall for copy number variants.If this option is selected, copy number variant calling will be carried out with either Spectre (default) or QDNAseq. To use QDNAseq instead of Spectre, use the option --use_qdnaseq. Spectre is only compatible with genome build hg38, and if QDNAseq is used, it is only compatible with genome builds hg37 and hg38.False
strbooleanEnable Straglr to genotype STR expansions.If this option is selected, genotyping of STR expansions will be carried out using Straglr. This sub-workflow is only compatible with genome build hg38.False
modbooleanEnable output of modified calls to a bedMethyl file [requires input BAM with Ml and Mm tags]This option is automatically selected and aggregation of modified calls with be carried out using modkit if Ml and Mm tags are found. Disable this option to prevent output of a bedMethyl file.False

Main options

Nextflow parameter nameTypeDescriptionHelpDefault
sample_namestringSample name to be displayed in workflow outputs.SAMPLE
bamstringBAM or unaligned BAM (uBAM) files for the sample to use in the analysis.This accepts one of two cases: (i) the path to a single BAM file; (ii) the path to a top-level directory containing BAM files. A sample name can be supplied with --sample.
refstringPath to a reference FASTA file.Reference against which to compare reads for variant calling.
basecaller_cfgstringName of the model to use for selecting a small variant calling model.Required for small variant calling. The basecaller configuration is used to automatically select the appropriate small variant calling model. The model list shows all models that are compatible for small variant calling with this workflow. You should select ‘custom’ to override the basecaller_cfg with clair3_model_path.dna_r10.4.1_e8.2_400bps_sup@v4.1.0
bam_min_coveragenumberMinimum read coverage required to run analysis.20
bedstringAn optional BED file enumerating regions to process for variant calling.
annotationbooleanSnpEff annotation.If this option is unselected, VCFs will not be annotated with SnpEff.True
phasedbooleanPerform phasing.This option enables phasing of SV, SNP and modifications, depending on which sub-workflow has been chosen; see README for more details.False
include_all_ctgsbooleanCall for variants on all sequences in the reference, otherwise small and structural variants will only be called on chr{1..22,X,Y,MT}.Enabling this option will call for variants on all contigs of the input reference sequence. Typically this option is not required as standard human reference sequences contain decoy and unplaced contigs that are usually omitted for the purpose of variant calling. This option might be useful for non-standard reference sequence databases.False
out_dirstringDirectory for output of all workflow results.output

Structural variant calling options

Nextflow parameter nameTypeDescriptionHelpDefault
tr_bedstringInput BED file containing tandem repeat annotations for the reference genome.Providing a tandem repeat BED can improve calling in repetitive regions. An appropriate tandem repeat BED can be downloaded for your reference genome from the Sniffles2 repository.

Structural variant benchmarking options

Nextflow parameter nameTypeDescriptionHelpDefault
sv_benchmarkbooleanBenchmark called structural variants.If this option is selected, automated benchmarking of structural variant calls will be carried out using Truvari.False

Copy number variant calling options

Nextflow parameter nameTypeDescriptionHelpDefault
use_qdnaseqbooleanUse QDNAseq for CNV calling.Set this to true to use QDNASeq for CNV calling instead of Spectre. QDNAseq is better suited to shorter reads such as those generated from adaptive sampling experiments.False
qdnaseq_bin_sizeintegerBin size for QDNAseq in kbp.Pre-computed bin annotations are available for a range of bin sizes. Larger sizes reduce noise, however this may result in reduced sensitivity.500

Modified base calling options

Nextflow parameter nameTypeDescriptionHelpDefault
force_strandbooleanRequire modkit to call strand-aware modifications.By default strand calls are collapsed (strand reported as ’.’). Enabling this will force stranding to be considered when calling modifications, creating one output per modification per strand and the report will be tabulated by both modification and strand.False

Short tandem repeat expansion genotyping options

Nextflow parameter nameTypeDescriptionHelpDefault
sexstringSex (male or female) to be passed to Straglr-genotype.The sex determines how many calls will be obtained for all repeats on chrX. Defaults to female if not specified.female

Advanced Options

Nextflow parameter nameTypeDescriptionHelpDefault
depth_intervalsbooleanOutput a bedGraph file with entries for each genomic interval featuring homogeneous depth.The output bedGraph file will have an entry for each genomic interval in which all positions have the same alignment depth. By default this workflow outputs summary depth information from your aligned reads. Per-base depth outputs are slower to generate but may be required for some downstream applications.False
GVCFbooleanEnable to output a gVCF file in addition to the VCF outputs (experimental).By default the the workflow outputs a VCF file containing only records where a variant has been detected. Enabling this option will output additionally a gVCF with records spanning all reference positions regardless of whether a variant was detected in the sample.False
downsample_coveragebooleanDownsample the coverage to along the genome.This options will trigger a downsampling of the read alignments to the target coverage specified by —downsample_coverage_target. Downsampling will make the workflow run faster but could lead to non-deterministic variant calls.False
downsample_coverage_targetnumberAverage coverage or reads to use for the analyses.This options will set the target coverage for the downsampling stage, if downsampling has been enabled.60

Multiprocessing Options

Nextflow parameter nameTypeDescriptionHelpDefault
threadsintegerSet max number of threads to use for more intense processes (limited by config executor cpus)4
ubam_map_threadsintegerSet max number of threads to use for aligning reads from uBAM (limited by config executor cpus)8
ubam_sort_threadsintegerSet max number of threads to use for sorting and indexing aligned reads from uBAM (limited by config executor cpus)3
ubam_bam2fq_threadsintegerSet max number of threads to use for uncompressing uBAM and generating FASTQ for alignment (limited by config executor cpus)1
merge_threadsintegerSet max number of threads to use for merging alignment files (limited by config executor cpus)4
modkit_threadsintegerTotal number of threads to use in modkit modified base calling (limited by config executor cpus)4


Output files may be aggregated including information for all samples or provided per sample. Per-sample files will be prefixed with respective aliases and represented below as {{ alias }}.

TitleFile pathDescriptionPer sample or aggregated
Report of the alignment statistics{{ alias }}.wf-human-alignment-report.htmlReport summarising the results of the alignment statistics for the sample.per-sample
Report of the SNP workflow{{ alias }}.wf-human-snp-report.htmlReport summarising the results of the SNP subworkflow for the sample.per-sample
Report of the SV workflow{{ alias }}.wf-human-sv-report.htmlReport summarising the results of the SV subworkflow for the sample.per-sample
Report of the CNV workflow{{ alias }}.wf-human-cnv-report.htmlReport summarising the results of the CNV subworkflow for the sample.per-sample
Report of the STR workflow{{ alias }}.wf-human-str-report.htmlReport summarising the results of the short tandem repeat subworkflow for the sample.per-sample
Short variant VCF{{ alias }}.wf_snp.vcf.gzVCF file with the SNPs for the sample.per-sample
Structural variant VCF{{ alias }}.wf_sv.vcf.gzVCF file with the SVs for the sample.per-sample
SNP and SV phased VCF{{ alias }}.wf_human_variation.phased.vcf.gzVCF file with the jointly phased SNPs and SVs for the sample.per-sample
Copy number variants VCF{{ alias }}.wf_cnv.vcf.gzVCF file with the CNV for the sample.per-sample
Modified bases BEDMethyl{{ alias }}.wf_mods.bedmethyl.gzBED file with the aggregated modification counts for the sample.per-sample
Short tandem repeat VCF{{ alias }}.wf_str.vcf.gzVCF file with the STR sites for the sample.per-sample
Alignment file{{ alias }}.cramCRAM or BAM file with the aligned reads for the sample, generated when the input file is unaligned.per-sample
Alignment file index{{ alias }}.cram.craiThe index of the resulting CRAM or BAM file with the reads for the sample, generated when the input file is unaligned.per-sample
Haplotagged alignment file{{ alias }}.haplotagged.cramCRAM or BAM file with the haplotagged reads for the sample.per-sample
Haplotagged alignment file index{{ alias }}.haplotagged.cram.craiThe index of the resulting CRAM or BAM file with the haplotagged reads for the sample.per-sample
Mean coverage for each region{{ alias }}.regions.bed.gzThe mean coverage in the individual regions of the genome in BED format.per-sample
Coverage per region above the given thresholds{{ alias }}.thresholds.bed.gzThe BED reporting the number of bases in each region that are covered at or above each threshold values (1x, 10x, 20x and 30x).per-sample
Distribution of the proportion of total bases covered by a given coverage value{{ alias }} cumulative distribution indicating the proportion of total bases covered by a given coverage value, both genome-wide and by sequence.per-sample
Mean coverage per sequence and target region{{ alias }}.mosdepth.summary.txtThe summary of mean depths per chromosome and within specified regions per chromosome.per-sample
BEDgraph of the single-base coverage{{ alias }}.per-base.bedgraph.gzThe single-base coverage of the genome in BED graph format.per-sample

Pipeline overview

The workflow is composed of 6 distinct subworkflows, each enabled by a command line option:

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

1. Input and data preparation

The workflow relies on two primary input files:

  1. A reference genome in FASTA format
  2. Sequencing data for the sample in the form of a single BAM file or a folder of BAM files, either aligned or unaligned.

When analysing human data, we recommend using human_g1k_v37.fasta.gz (FTP link) or GCA_000001405.15_GRCh38_no_alt_analysis_set.fna.gz (FTP link). For more information see this blog post which outlines potential pitfalls with the various flavours of human references.

The input BAM file can be generated using the wf-basecalling workflow, which is up to date with the current dorado releases and models.

2. Data QC and pre-processing

The workflow starts by performing multiple checks of the input BAM file, as well as computing:

  1. depth of sequencing with mosdepth;
  2. read alignment statistics with fastcat.

After computing the coverage, the workflow will check that the input BAM file has a depth greater than --bam_min_coverage. In case the user specify --bam_min_coverage 0, the check will be skipped and the workflow will proceed directly to the downstream analyses. Some components work better withing certain ranges of coverage, and the user might achieve better results by providing a target coverage to downsample to. The user can set --downsample_coverage true to enable the downsampling of the reads, and --downsample_coverage_target {{ X }} to specify the target coverage (default: 60x).

3. Small variant calling with Clair3

The workflow implements a deconstructed version of Clair3 (v1.0.4) to call germline variants. The appropriate model can be provided with the --basecaller_cfg option. To decide on the appropriate model you can check out the Dorado documentation for a list of available basecalling models. This workflow takes advantage of the parallel nature of Nextflow, providing optimal efficiency in high-performance, distributed systems. The workflow will automatically call small variants (SNPs and indels), collect statistics, annotate them with SnpEff (and additionally for SNPs, ClinVar details), and create a report summarising the findings.

If desired, the workflow can perform phasing of structural variants by using the --phased option. This will lead the workflow to use longphase to perform phasing of the variants, with the option to use whatshap instead by setting --use_longphase false. Deactivating the longphase phasing will not disable the final joint phasing with longphase, and if you want the individually phased VCFs you should provide the --output_separate_phased option. The phasing will also generate a GFF file with the annotation of the phase blocks, facilitating the detection of these within genome visualizers.

4. Structural variant (SV) calling with Sniffles2

The workflow allows for calling of SVs using long-read sequencing data with Sniffles2. The workflow will perform SV calling, filtering and generation of a report. Optionally, the workflow can also evaluate calls on HG002 against a truth set (provided the input data was aligned to HG19). The SV workflow takes an optional --tr_bed option to specify tandem repeats in the reference sequence --- see the sniffles documentation for more information. SVs can be phased using --phased. However, this will cause the workflow to run SNP analysis, as SV phasing relies on the haplotagged reads generated in this stage.

5. Modified base calling with modkit

Modified base calling can be performed by specifying --mod. The workflow will call modified bases using modkit. The workflow will automatically check whether the files contain the appropriate MM/ML tags, required for running modkit pileup. If the tags are not found, the workflow will not run the individual analysis, but will still run the other subworkflows requested by the user. The default behaviour of the workflow is to run modkit with the --cpg --combine-strands options set. It is possible to report strand-aware modifications by providing --force_strand, which will trigger modkit to run in default mode. The resulting bedMethyl will include modifications for each site on each strand separately. The modkit run can be fully customized by providing --modkit_args. This will override any preset, and allow full control over the run of modkit. Haplotype-resolved aggregated counts of modified bases can be obtained with the --phased option. This will generate three distinct BEDMethyl files with the naming pattern {{ alias }}_{{ haplotype }}.wf_mods.bedmethyl.gz, where haplotype can be 1, 2 or ungrouped.

6a. Copy number variants (CNV) calling with Spectre

CNV calling is performed using a fork of Spectre, using the --cnv flag. Spectre is the default CNV caller in the workflow, and is compatible with hg38/GRCh38. The output of this workflow is a VCF of CNV calls, annotated with SnpEff.

6b. Copy number variants (CNV) calling with QDNASeq

CNV calling may alternatively be performed using QDNAseq, using --cnv --use_qdnaseq. This workflow is compatible with genome builds hg19/GRCh37 or hg38/GRCh38, and is recommended for shallow WGS or adaptive sampling data. In addition to the VCF of CNV calls, the workflow emits QDNAseq-generated plots and BED files of both raw read counts per bin and corrected, normalised, and smoothed read counts per bin. Please note that QDNAseq was the default CNV caller until version 1.11.0 of the workflow, and the additional --use_qdnaseq flag is now required to use it.

7. Short tandem repeat (STR) genotyping with Straglr

STR genotyping is performed using a fork of straglr. This workflow is compatible with genome build hg38/GRCh38. 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. In addition to a gzipped VCF file containing STRs found in the dataset, the workflow emits a TSV straglr output containing reads spanning STRs, and a haplotagged BAM.

8. Phasing variants

Variant phasing is switched on simply using the --phased option. By default, the workflow uses longphase to perform phasing of the variants, with the option to use whatshap instead by setting --use_longphase false. The workflow will automatically turn on the necessary phasing processes based on the selected subworkflows. The behaviour of the phasing is summarised in the below table:

Phased SNP VCFPhased SV VCFJoint SV+SNP phased VCFPhased bedMethyl

The joint physical phasing of SNP and SVs can only be performed with longphase by selecting the options: --phased --snp --sv. Setting --use_longphase false will not disable the final joint phasing with longphase.

In some circumstances, users may wish to keep the separate VCF files before joint phasing. This can be done with --output_separate_phased.

Running the phasing is a compute intensive process. Running the workflow in phasing mode doubles the runtime, and significantly increases the storage requirements to the order of terabytes.

9. Variant annotation

Annotation will be performed automatically by the SNP and SV subworkflows, and can be disabled by the user with --annotation false. The workflow will annotate the variants using SnpEff, and currently only support the human hg19 and hg38 genomes. Additionally, the workflow will add the ClinVar annotations for the SNP variants.

Running the workflow on non-human samples will require this option to be disabled. For more detail, see Section 10 below.

10. Genome compatibility and running the workflow on non-human genomes

Some of the sub-workflows in wf-human-variation are restricted to certain genome builds, which means they will not be executable on non-human genomes or human genome builds outside hg19/GRCh37 and hg38/GRCh38. The following table summarises which subworkflows and options are available (or required) for a desired input genome:

Genome--snp--sv--mod--cnv--cnv --use_qdnaseq--str--annotation false--include_all_ctgs
Other human
Non human

* As noted above, annotation is performed by default but can be switched off for hg19/GRCh37 and hg38/GRCh38.


  • Annotations for --snp and --sv are generated using SnpEff. For --snp, additional ClinVar annotations are displayed in the report where available (please note, the report will not display any variants classified as ‘Benign’ or ‘Likely benign’, however these variants will be present in the output VCF).
  • Specifying a suitable tandem repeat BED for your reference with --tr_bed can improve the accuracy of SV calling.
  • Aggregation of modified calls with --mod requires data to be basecalled with a model that includes base modifications, providing the MM and ML BAM tags
  • CRAM files generated within the workflow cannot be read without the corresponding reference
  • The STR workflow performs genotyping of specific repeats, which can be found here.
  • By default, SNPs and SVs will be called on chromosomes 1-22, X, Y and MT; to call sites on other sequences enable the --include_all_ctgs option.
  • While designed to work on human genomes, the workflow can be run on non-human species by setting --cnv false --str false --annotation false --include_all_ctgs true.
  • Ensure that the provided reference and BED files use the same chromosome coding (for example, that they both have the chr prefix, or they both to not have it).
  • If unaligned reads were provided, the workflow will output a CRAM file (or BAM if the user runs the --cnv option) containing the alignments used to make the downstream variant calls


If your question is not answered here, please report any issues or suggestions on the github issues page or start a discussion on the community.

See the EPI2ME website for lots of other resources and blog posts.




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