Introduction

sanger-tol/variantcalling is a bioinformatics best-practice analysis pipeline for variant calling using DeepVariant for PacBio data.

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 LSF infrastructure. This ensures that the pipeline runs on LSF, 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.

Pipeline summary

The pipeline takes aligned or unaligned PacBio sample reads (CRAM/BAM files) from a CSV file and the reference file in FASTA format, and then uses DeepVariant tool to make variant calling.

Steps involved:

• Split fasta file into smaller files, normally one sequence per file unless the sequences are too small.
• Align the reads if not aligned.
• Merge input BAM/CRAM files together if they have the same sample names.
• Filter out reads using the -F 0x900 option to only retain the primary alignments.
• Run DeepVariant using filtered BAM/CRAM files against each of split fasta files.
• Merge all VCF and GVCF files generated by DeepVariant by sample together for each input BAM/CRAM file.
• Run VCFtools to calculate heterozygosity and per site nucleotide diversity.

Quick Start

1. Install Nextflow (>=23.10.1)

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:

```bash # for aligned reads nextflow run sanger-tol/variantcalling -profile test,YOURPROFILE --outdir

# for unaligned reads nextflow run sanger-tol/variantcalling -profile test_align,YOURPROFILE --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.

1. Start running your own analysis!

bash nextflow run sanger-tol/variantcalling --input samplesheet.csv --outdir <OUTDIR> --fasta genome.fasta.gz -profile <docker/singularity/podman/shifter/charliecloud/conda/institute>

Credits

sanger-tol/variantcalling was originally written by Guoying Qi.

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

• Priyanka Surana for the pipeline design, code review and Nextflow technical supports.
• Matthieu Muffato for the pipeline design, code review and Nextflow technical supports.

We also acknowledge the work of Damon-Lee Pointon for attempting to create an short read variant calling subworkflow.

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 #pipelines channel. Please create an issue on GitHub if you are not on the Sanger slack channel.

Citations

If you use sanger-tol/variantcalling for your analysis, please cite it using the following doi: 10.5281/zenodo.7890527

An extensive list of references for the tools used by the pipeline can be found in the CITATIONS.md file.

This pipeline uses code and infrastructure developed and maintained by the nf-core community, reused here under the MIT license.

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.