nf-core/variantprioritization

Edit

Introduction

nf-core/variantprioritization consumes small variant calls (SNVs/InDels) in VCF format and optionally copy number alteration (CNA) segments, and produces annotated reports using PCGR.

The pipeline is designed to work well with outputs from nf-core/sarek (e.g. Mutect2, Strelka), but any compatible VCFs can be used.

Samplesheet input

You will need to create a samplesheet with information about the samples you would like to analyse before running the pipeline. Use the --input parameter to specify its location.

It must be a comma-separated file with a header row and the following required columns:

• patient: Patient / subject identifier (no spaces)
• status: 0 = normal / germline, 1 = tumor / somatic
• sample: Sample identifier (no spaces)
• vcf: Path to a VCF / VCF.GZ file

Optionally, you can provide:

• cna: Path to a CNA segments file (required for somatic rows when --cna_analysis is enabled)

--input '[path to samplesheet file]'

Multiple VCFs per sample

If you have multiple VCFs for the same sample (e.g. one per caller), provide one row per VCF. The pipeline will keep caller-specific files separate during preprocessing and then consolidate/intersect somatic calls per sample for PCGR reporting.

CNA files

If you run with --cna_analysis, CNA segments are required for somatic (status=1) rows. The CNA file is expected to be either ASCAT-like or CNVkit-like and will be reformatted into the allele-specific schema required by PCGR.

VCF filename conventions (caller detection)

The pipeline extracts the caller name from the VCF filename by splitting on dots (.) and taking the second field (e.g. *.mutect2.*.vcf.gz). For Strelka, it expects strelka.somatic_snvs or strelka.somatic_indels to appear as the second and third fields.

This convention matches typical nf-core/sarek outputs, for example:

• HCC1395T_vs_HCC1395N.mutect2.vcf.gz
• HCC1395T_vs_HCC1395N.strelka.somatic_snvs.vcf.gz

Example samplesheet

The workflow accepts as input a samplesheet.csv file containing the paths to SNV/InDel VCF files and ASCAT copy number abberation files. We have efforted to mimick the samplesheet specifications of nf-core/sarek for ease of use:

An example of a valid samplesheet is given below:

patient,status,sample,vcf,cna
HCC1395,1,HCC1395T,HCC1395T_vs_HCC1395N.mutect2.vcf.gz,HCC1395T.segments.txt
HCC1395,1,HCC1395T,HCC1395T_vs_HCC1395N.freebayes.vcf.gz,HCC1395T.segments.txt
HCC1395,1,HCC1395T,HCC1395T_vs_HCC1395N.strelka.somatic_snvs.vcf.gz,HCC1395T.segments.txt
HCC1395,1,HCC1395T,HCC1395T_vs_HCC1395N.strelka.somatic_indels.vcf.gz,HCC1395T.segments.txt
HCC1395,0,HCC1395N,HCC1395N.deepvariant.vcf.gz,
HCC1395,0,HCC1395N,HCC1395N.haplotypecaller.vcf.gz,
HCC1396,1,HCC1396T,HCC1396T_vs_HCC1396N.mutect2.vcf.gz,
HCC1396,1,HCC1396T,HCC1396T_vs_HCC1396N.strelka.somatic_snvs.vcf.gz,
HCC1396,1,HCC1396T,HCC1396T_vs_HCC1396N.strelka.somatic_indels.vcf.gz,

An example samplesheet is provided in assets/samplesheet.csv.

copy number abberation files must be present for every sample entry when --cna_analysis true.

Running the pipeline

The typical command for running the pipeline is as follows:

nextflow run nf-core/variantprioritization \
  -profile <docker/singularity/.../institute> \
  --input ./samplesheet.csv \
  --outdir ./results \
  --genome GRCh38

Note that the pipeline will create the following files in your working directory:

work                # Directory containing the nextflow working files
<OUTDIR>            # Finished results in specified location (defined with --outdir)
.nextflow_log       # Log file from Nextflow
# Other nextflow hidden files, eg. history of pipeline runs and old logs.

If you wish to repeatedly use the same parameters for multiple runs, rather than specifying each flag in the command, you can specify these in a params file.

For more details and further functionality, please refer to the usage documentation and the parameter documentation.

Updating the pipeline

When you run the above command, Nextflow automatically pulls the pipeline code from GitHub and stores it as a cached version. When running the pipeline after this, it will always use the cached version if available - even if the pipeline has been updated since. To make sure that you’re running the latest version of the pipeline, make sure that you regularly update the cached version of the pipeline:

nextflow pull nf-core/variantprioritization

Reproducibility

It is a good idea to specify the pipeline version when running the pipeline on your data. This ensures that a specific version of the pipeline code and software are used when you run your pipeline. If you keep using the same tag, you’ll be running the same version of the pipeline, even if there have been changes to the code since.

First, go to the nf-core/variantprioritization releases page and find the latest pipeline version - numeric only (eg. 1.3.1). Then specify this when running the pipeline with -r (one hyphen) - eg. -r 1.3.1. Of course, you can switch to another version by changing the number after the -r flag.

This version number will be logged in reports when you run the pipeline, so that you’ll know what you used when you look back in the future. For example, at the bottom of the MultiQC reports.

To further assist in reproducibility, you can use share and reuse parameter files to repeat pipeline runs with the same settings without having to write out a command with every single parameter.

Variant consolidation

Somatic variants called by multiple tools are reformatted to match PCGR specifications making them easily searchable in the HTML ouput.

Tumor sample depth (TDP), allele frequency (TAF) and allelic depths for the ref and alt (ADT) are manually calculated and when applicable, applied to the normal sample (NDP, NAF, ADN):

HCC1395T_vs_HCC1395N.mutect2.vcf.gz
#CHROM  POS     ID      REF     ALT     QUAL    FILTER  INFO    FORMAT  HCC1395_HCC1395N        HCC1395_HCC1395T
chr1    1212740 .       A       C       .       PASS    AS_SB_TABLE=63,80|49,76;DP=282;ECNT=1;MBQ=20,20;MFRL=151,154;MMQ=60,60;MPOS=30;NALOD=1.94;NLOD=25.89;POPAF=6.00;TLOD=341.76     GT:AD:AF:DP:F1R2:F2R1:FAD:SB 0/0:143,0:0.011:143:36,0:36,0:86,0:63,80,0,0    0/1:0,125:0.988:125:0,28:0,37:0,78:0,0,49,76

TDP=125;NDP=143;TAF=0.988;NAF=0.011;ADT=0,125;ADN=143,0;TAL=mutect2

HCC1395T_vs_HCC1395N.strelka.somatic_snvs.vcf.gz
#CHROM  POS     ID      REF     ALT     QUAL    FILTER  INFO    FORMAT  NORMAL  TUMOR
chr1    1212740 .       A       C       .       PASS    DP=271;MQ=60.00;MQ0=0;NT=ref;QSS=790;QSS_NT=3070;ReadPosRankSum=0.00;SGT=AA->AC;SNVSB=0.00;SOMATIC;SomaticEVS=19.73;TQSS=1;TQSS_NT=1    DP:FDP:SDP:SUBDP:AU:CU:GU:TU 145:0:0:0:145,145:0,0:0,0:0,0   126:0:0:0:0,0:126,126:0,0:0,0

TDP=126;NDP=145;TAF=1;NAF=0;ADT=0,126;ADN=145,0;TAL=strelka

Finally, the maximum values for TAF, TDP, NAF, NDP, ADT, ADN are taken as outputs for the consolidate variant call. In addition, values present in the ID and QUAL column (i.e not '.') are reported if present in any of the original calls:

1       1212740 .       A       C       3793.8  PASS    NDP=145;NAF=0.011;TDP=126;TAF=1;TAL=mutect2,strelka

Core Nextflow arguments

-profile

Use this parameter to choose a configuration profile. Profiles can give configuration presets for different compute environments.

Several generic profiles are bundled with the pipeline which instruct the pipeline to use software packaged using different methods (Docker, Singularity, Podman, Shifter, Charliecloud, Apptainer, Conda) - see below.

The pipeline also dynamically loads configurations from https://github.com/nf-core/configs when it runs, making multiple config profiles for various institutional clusters available at run time. For more information and to check if your system is supported, please see the nf-core/configs documentation.

Note that multiple profiles can be loaded, for example: -profile test,docker - the order of arguments is important! They are loaded in sequence, so later profiles can overwrite earlier profiles.

If -profile is not specified, the pipeline will run locally and expect all software to be installed and available on the PATH. This is not recommended, since it can lead to different results on different machines dependent on the computer environment.

• test
  • A profile with a complete configuration for automated testing
  • Includes links to test data so needs no other parameters
• docker
  • A generic configuration profile to be used with Docker
• singularity
  • A generic configuration profile to be used with Singularity
• podman
  • A generic configuration profile to be used with Podman
• shifter
  • A generic configuration profile to be used with Shifter
• charliecloud
  • A generic configuration profile to be used with Charliecloud
• apptainer
  • A generic configuration profile to be used with Apptainer
• wave
  • A generic configuration profile to enable Wave containers. Use together with one of the above (requires Nextflow 24.03.0-edge or later).
• conda
  • A generic configuration profile to be used with Conda. Please only use Conda as a last resort i.e. when it’s not possible to run the pipeline with Docker, Singularity, Podman, Shifter, Charliecloud, or Apptainer.

-resume

Specify this when restarting a pipeline. Nextflow will use cached results from any pipeline steps where the inputs are the same, continuing from where it got to previously. For input to be considered the same, not only the names must be identical but the files’ contents as well. For more info about this parameter, see this blog post.

You can also supply a run name to resume a specific run: -resume [run-name]. Use the nextflow log command to show previous run names.

-c

Specify the path to a specific config file (this is a core Nextflow command). See the nf-core website documentation for more information.

Custom configuration

Resource requests

Whilst the default requirements set within the pipeline will hopefully work for most people and with most input data, you may find that you want to customise the compute resources that the pipeline requests. Each step in the pipeline has a default set of requirements for number of CPUs, memory and time. For most of the pipeline steps, if the job exits with any of the error codes specified here it will automatically be resubmitted with higher resources request (2 x original, then 3 x original). If it still fails after the third attempt then the pipeline execution is stopped.

To change the resource requests, please see the max resources and tuning workflow resources section of the nf-core website.

Custom Containers

In some cases, you may wish to change the container or conda environment used by a pipeline steps for a particular tool. By default, nf-core pipelines use containers and software from the biocontainers or bioconda projects. However, in some cases the pipeline specified version maybe out of date.

To use a different container from the default container or conda environment specified in a pipeline, please see the updating tool versions section of the nf-core website.

Custom Tool Arguments

A pipeline might not always support every possible argument or option of a particular tool used in pipeline. Fortunately, nf-core pipelines provide some freedom to users to insert additional parameters that the pipeline does not include by default.

To learn how to provide additional arguments to a particular tool of the pipeline, please see the customising tool arguments section of the nf-core website.

nf-core/configs

In most cases, you will only need to create a custom config as a one-off but if you and others within your organisation are likely to be running nf-core pipelines regularly and need to use the same settings regularly it may be a good idea to request that your custom config file is uploaded to the nf-core/configs git repository. Before you do this please can you test that the config file works with your pipeline of choice using the -c parameter. You can then create a pull request to the nf-core/configs repository with the addition of your config file, associated documentation file (see examples in nf-core/configs/docs), and amending nfcore_custom.config to include your custom profile.

See the main Nextflow documentation for more information about creating your own configuration files.

If you have any questions or issues please send us a message on Slack on the #configs channel.

Running in the background

Nextflow handles job submissions and supervises the running jobs. The Nextflow process must run until the pipeline is finished.

The Nextflow -bg flag launches Nextflow in the background, detached from your terminal so that the workflow does not stop if you log out of your session. The logs are saved to a file.

Alternatively, you can use screen / tmux or similar tool to create a detached session which you can log back into at a later time. Some HPC setups also allow you to run nextflow within a cluster job submitted your job scheduler (from where it submits more jobs).

Nextflow memory requirements

In some cases, the Nextflow Java virtual machines can start to request a large amount of memory. We recommend adding the following line to your environment to limit this (typically in ~/.bashrc or ~./bash_profile):

NXF_OPTS='-Xms1g -Xmx4g'