Using code from scholl-lab/merge-multisample-vcf, merge the VCFs for each sample into a single VCF. This will be used for annotation.

wget https://raw.githubusercontent.com/scholl-lab/merge-multisample-vcf/main/merge_sequential.smk
wget https://raw.githubusercontent.com/scholl-lab/merge-multisample-vcf/main/run_merge_sequential.sh

Before running the pipeline to set up the config.yaml file as described in the scholl-lab/merge-multisample-vcf README.md file.

This will merge the VCFs for each sample into a single VCF. The resulting VCF all_merged.vcf.gz will be in the results/final directory.

sbatch run_merge_sequential.sh

Using the bcftools scatter plugin the merged VCF is split (100k variants per file) into smaller chunks for annotation. This will be used for annotation.

mkdir -p results/scatter/

This will split the VCF into smaller chunks to allow for parallel annotation. Bcftools was used in Version: 1.17 (using htslib 1.17) using the following command. it was installed using mamba with mamba create -n bcftools bcftools. Activate the conda environment containing bcftools.

Parameters are set as follows:

• +scatter - run the scatter plugin
• results/final/all_merged.vcf.gz - the input VCF
• -Oz - output a compressed VCF
• --threads 4 - use 4 threads
• -o results/scatter/ - output directory
• -n 100000 - number of variants per file
• -p all_merged. - prefix for the output files

conda activate bcftools
bcftools +scatter results/final/all_merged.vcf.gz -Oz --threads 4 -o results/scatter/ -n 100000 -p all_merged.

As the jo takes relatively long to run, it is recommended to submit it as a cluster job using the following command (helper script in scripts/bcftools_scatter.sh)):

sbatch bcftools_scatter.sh

You can also provide command line arguments to override the default parameter values of the script, for example:

sbatch scripts/bcftools_scatter.sh my_input.vcf.gz my_output_directory/ 50000 my_prefix.

Defaults are set as follows:

• INFILE=${1:-results/final/all_merged.vcf.gz}
• OUTDIR=${2:-results/scatter/}
• VARIANTS=${3:-100000}
• PREFIX=${4:-all_merged.}