Introduction

CIBERER/GdTBioinfo-nf-mosaicism is a bioinformatics best-practice analysis pipeline for detection of mosaicism through NGS 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! 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!

Pipeline summary

This pipeline uses two variant callers to detect low frequency variants, VarScan and VarDict. The result file is a VCF file obtained after intersecting the two VCF files.

1. VarScan (VarScan)

1.1. Sorting BAM file (Samtools)

1.2. Obtaining mpileup file (Samtools)

1.3. Variant Calling (VarScan)

1. VarDict (VarDictJava)

2.1. Variant Calling (VarDictJava)

1. Mutect2 (Mutect2)

3.1 Generate reference sequence dictionary (CreateSequenceDictionary)

3.2 Variant Calling (Mutect2)

1. Intersection of the three VCF files (Bedtools)

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:

console nextflow pull CIBERER/GdTBioinfo-nf-mosaicism nextflow run CIBERER/GdTBioinfo-nf-mosaicism -profile test,YOURPROFILE --outdir <OUTDIR>

Then, download the profile_test.md5 file, locate it in the root folder and run the next command:

console md5sum -c profile_test.md5

If the output is like this, everything is working fine!:

console ./results/join/Sample_zenodo_T1.vcf: OK ./results/mutect2/Sample_zenodo_T1.mutect2.vcf.gz: OK ./results/mutect2/Sample_zenodo_T1.mutect2.vcf.gz.stats: OK ./results/mutect2/Sample_zenodo_T1.mutect2.vcf.gz.tbi: OK ./results/vardictjava/Sample_zenodo_T1.mpileup: OK ./results/vardictjava/Sample_zenodo_T1.txt.gz: OK ./results/vardictjava/Sample_zenodo_T1..vcf.gz: OK ./results/varscan/Sample_zenodo_T1.v2.vcf.gz: OK

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!

<!-- TODO nf-core: Update the example "typical command" below used to run the pipeline -->

console nextflow run CIBERER/GdTBioinfo-nf-mosaicism --input path/to/samplesheet.csv --outdir <OUTDIR> --fasta path/to/reference/genome.fa --fai path/to/reference/genome.fa.fai --chrom_sizes path/to/referece/chrom.sizes --germline_resource path/to/population/germline_resource.vcf.gz --germline_resource_tbi path/to/population/germline_resource.vcf.gz.tbi -profile <docker/singularity/podman/shifter/charliecloud/conda/institute>

Documentation

The CIBERER-pipelines /mosaicism pipeline comes with documentation about the pipeline usage, parameters and output.

Credits

This pipeline was originally written by Carlos Ruiz, Marta Sevilla and Yolanda Benítez at the Centro de Investigaciones Biomédicas en Red de Enfermedades Raras, CIBERER.

Main authors:

• Carlos Ruiz
• Marta Sevilla
• Yolanda Benítez
• Pedro Garrido
• Paula Iborra

Contributions and Support

If you would like to contribute to this pipeline, please contact with:

carlos.ruiza@upf.edu, marta.sevilla@upf.edu or yolanda.benitez@ciberer.es

<!-- For further information or help, don't hesitate to get in touch on the Slack #mosaicism channel (you can join with this invite). -->

Citations

1. VarScan (VarScan)
2. Samtools (Samtools)
3. VarDictJava (VarDictJava)
4. Mutect2 (GATK)
5. Bedtools Bedtools
6. Tabix(Tabix)

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. In addition, references of tools and data used in this pipeline are as follows: