wi-gatk¶

The wi-gatk pipeline filters and calls variants from wild isolate sequence data.

Pipeline Overview¶


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    parameters                 description                           Set/Default
    ==========                 ===========                           ========================
    --debug                    Use --debug to indicate debug mode    null
    --output                   Release Directory                     WI-{date}
    --sample_sheet             Sample sheet                          null
    --bam_location             Directory of bam files                /projects/b1059/data/{species}/WI/alignments/
    --mito_name                Contig not to polarize hetero sites   MtDNA

    Reference Genome
    --------------- 
    --reference_base           Location of ref genomes               /projects/b1059/data/{species}/genomes/
    --species/project/build    These 4 params form --reference       {species} / {project} / {ws_build}

    Variant Filters         
    ---------------           
    --min_depth                Minimum variant depth                 5
    --qual                     Variant QUAL score                    30
    --strand_odds_ratio        SOR_strand_odds_ratio                 5
    --quality_by_depth         QD_quality_by_depth                   20
    --fisherstrand             FS_fisher_strand                      100
    --high_missing             Max % missing genotypes               0.95
    --high_heterozygosity      Max % max heterozygosity              0.10

Pipeline-overview

Software Requirements¶

The latest update requires Nextflow version 24+. On Rockfish, you can access this version by loading the nf24_env conda environment prior to running the pipeline command:

module load python/anaconda
source activate /data/eande106/software/conda_envs/nf24_env

Relevant Docker Images¶

andersenlab/gatk4 (link): Docker image is created within this pipeline using GitHub actions. Whenever a change is made to env/gatk4.Dockerfile or .github/workflows/build_docker.yml GitHub actions will create a new docker image and push if successful
andersenlab/r_packages (link): Docker image is created manually, code can be found in the dockerfile repo.

Important

Make sure that you have gone through the Nextflow setup before running this workflow.

Note

If you need to work with the docker container, you will need to create an interactive session as singularity can't be run on Rockfish login nodes.

interact -n1 -pexpress module load singularity singularity shell [--bind local_dir:container_dir] /vast/eande106/singularity/<image_name>

Usage¶

Note: if you are having issues running Nextflow or need reminders, check out the Nextflow page.

Testing on Rockfish¶

This command uses a test dataset

nextflow run -latest andersenlab/wi-gatk --debug

Running on Rockfish¶

You should run this in a screen or tmux session.

Note: if you are having issues running Nextflow or need reminders, check out the Nextflow page.

nextflow run -latest andersenlab/wi-gatk --sample_sheet <path_to_sample_sheet> --species c_elegans

Parameters¶

-profile¶

There are three configuration profiles for this pipeline.

rockfish - Used for running on Rockfish (default).
quest - Used for running on Quest.
local - Used for local development.

Note

If you forget to add a -profile, the rockfish profile will be chosen as default

--sample_sheet¶

The sample sheet is automatically generated from alignment-nf in the output folder under the name sample_sheet_for_seq_sheet_ALL.tsv. The sample sheet contains 5 columns as detailed below:

strain	bam	bai	coverage	percent_mapped
AB1	AB1.bam	AB1.bam.bai	64	99.4
AB4	AB4.bam	AB4.bam.bai	52	99.2
BRC20067	BRC20067.bam	BRC20067.bam.bai	30	92.5

Important

It is essential that you always use the pipelines and scripts to generate this sample sheet and NEVER manually. There are lots of strains and we want to make sure the entire process can be reproduced.

Note

The sample sheet produced from alignment-nf is only for strains that you ran in the alignment pipeline most recently. If you want to combine old strains with new strains, you will have to combine two or more sample sheets. If you are running a species-wide analysis for CaeNDR, please follow the notes in the full WI protocol here

--bam_location (optional)¶

Path to directory holding all the alignment files for strains in the analysis. Defaults to /vast/eande106/data/{species}/WI/alignments/

Important

Remember to move your bam files output from alignment-nf to this location prior to running wi-gatk. In most cases, you will want to run wi-gatk on all samples, new and old combined.

--species (optional)¶

default = c_elegans

Options: c_elegans, c_briggsae, or c_tropicalis

--project (optional)¶

default = PRJNA13758

WormBase project ID for selected species. Choose from some examples here

--ws_build (optional)¶

default = WS283

WormBase version to use for reference genome.

--reference (optional)¶

A fasta reference indexed with BWA. On Rockfish, the reference is available here:

/vast/eande106/data/c_elegans/genomes/PRJNA13758/WS283/c_elegans.PRJNA13758.WS283.genome.fa.gz

Note

If running on Rockfish, instead of changing the reference parameter, opt to change the species, project, and ws_build for other species like c_briggsae (and then the reference will change automatically)

--mito_name (optional)¶

Name of contig to skip het polarization. Might need to change for other species besides c_elegans if the mitochondria contig is named differently. Defaults to MtDNA.

--output (optional)¶

A directory in which to output results. By default it will be WI-YYYYMMDD where YYYYMMDD is todays date.

Output¶

The final output directory looks like this:


├── variation
│   ├── *.hard-filter.vcf.gz
│   ├── *.hard-filter.vcf.tbi
│   ├── *.hard-filter.stats.txt
│   ├── *.hard-filter.filter_stats.txt
│   ├── *.soft-filter.vcf.gz
│   ├── *.soft-filter.vcf.tbi
│   ├── *.soft-filter.stats.txt
│   └── *.soft-filter.filter_stats.txt
└── report
    ├── multiqc.html
    └── multiqc_data
        └── multiqc_*.json

Data Storage¶

Cleanup¶

The hard-filter.vcf is the input for both the concordance-nf pipeline and the post-gatk-nf pipeline. Once both pipelines have been completed successfully, the hard and soft filter vcf and index files (everything output in the variation folder) can be moved to /vast/eande106/data/{species}/WI/variation/{date}/vcf.