Introduction:

MitoH3 is a pipeline that calls MT variants from sequence file (cram file). In addition to the regular MT reference sequence file, the pipeline also applied a shifted MT reference file to get calls at D-loop region more precisely.

There are two main steps of the pipeline:

• 1. Generate raw vcf file from cram file.
• 2. Call haplogroup from cleaned vcf file; Split raw vcf file into homoplasmic variants vcf and heteroplasmic variants vcf at a given alternative allele frequency cutoff.

1. Preparation:

• build singularity image using definition file:

How to set up singularity remote build

wget https://raw.githubusercontent.com/MarchOnion/MitoH3/main/MitoH3.def

singularity build --remote MitoH3.sif MitoH3.def

• download reference file and prepare json file:

original file link

wget https://www.dropbox.com/s/4u1mz7h7ws1z89k/MT_WDL_ref.zip

unzip MT_WDL_ref.zip

• 1KG project cram file example:

wget ftp://ftp.sra.ebi.ac.uk/vol1/run/ERR398/ERR3988882/HG01433.final.cram

wget ftp://ftp.sra.ebi.ac.uk/vol1/run/ERR398/ERR3988882/HG01433.final.cram.crai

• prepare json file:

wget https://raw.githubusercontent.com/MarchOnion/MitoH3/main/input.json

dir=`pwd`

sed -i "s|your_local_path|${dir}|g" input.json 

2. Variants calling

• step1:

singularity exec --bind "$dir" MitoH3.sif bash /script/run1.sh input.json

• step2:

step1_output=cromwell-executions/MitochondriaPipeline/*/call-SplitMultiAllelicSites/execution/*.final.split.vcf

singularity exec --bind "$dir" MitoH3.sif  bash /script/run2.sh   $step1_output    prefix  0.05 0.95

Supplementary

• example script for merge vcf files is also provided.

https://raw.githubusercontent.com/MarchOnion/MitoH3/main/merge_vcf_files.sh

• reference pipeline