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CHIP pipeline

Setup

This pipeline is for calling LCHIP and MCHIP from WES/WGS CRAM files. The pipeline has 3 steps in total:

Before STEP1, it is recommended to download MitoHPC (https://github.com/ArkingLab/MitoHPC) and run init.sh so the required helper scripts and reference setup are available for this pipeline.

In order to run the full CHIP pipeline, please apply the following changes to scripts within MitoHPC:

  1. fixmutect2Vcf.pl: comment out line 46 and 47
  2. filterVcf.pl: comment out line 91, add the following immediately after:
    if($F[7] eq "." || $F[7] eq "") {
        $F[7] = "SM=$opt{sample}";
    } else {
        $F[7] .= ";SM=$opt{sample}";
    }

and comment out line 115 and 116

Running full MCHIP and LCHIP pipeline

STEP1

Step1 is running Mutect2 for CHIP genes, to call from CHIP regions

  • For this step, first run the download script to download all files, then run mutect2.sh script to call CHIP variants.
  • After mutect2.sh has finished for the cohort, run summary.sh to pull the per-sample calls into the summary VCF used as input for STEP2.
  • Run both MCHIP and LCHIP

STEP2

STEP2_LCHIP

  • Copy the mutect2.lchip.filter.1.vcf file from step 1 to this directory.
  • Remove header: tail -n +32 mutect2.lchip.filter.1.vcf > mutect2.lchip.filternh.1.vcf
  • Insert a header row: (echo -e "#CHROM\tPOS\tID\tREF\tALT\tQUAL\tFILTER\tINFO\tFORMAT\tSAMPLE"; cat mutect2.lchip.filternh.1.vcf) > mutect2.lchip.filterh.1.vcf
  • Copy and rename the input file: mv mutect2.lchip.filterh.1.vcf Input.vcf
  • Make sure the following packages are installed under command line R:
library("dplyr")
library("stringr")
library("data.table")
PathFilt

Download annovar:

wget www.openbioinformatics.org/annovar/download/0wgxR2rIVP/annovar.latest.tar.gz
tar -zxvf annovar.latest.tar.gz
cd annovar
chmod +x *.pl
perl annotate_variation.pl -buildver hg38 -downdb -webfrom annovar refGene humandb/
perl annotate_variation.pl -buildver hg38 -downdb -webfrom annovar gnomad211_exome humandb/
  • Change permission for all files: chmod +x *
  • Copy file over: cp ../Input.vcf ./
  • Run START bash script first: . ./Manual_START_UKB_Filter.sh
  • Then the Rscript: Rscript Manual_UKB_FinalStretch.R
PutativeFilt
cp ../Input.vcf ./
chmod +x *

For putativeFilt, the script needs sex information for each sample, where column id is sample id and column sex is men/women, and save as idsex.txt.

  • Run START script first: . ./Manual_START_UKB_Filter.sh
  • Then the Rscript: Rscript Manual_UKB_FinalStretch.R
Merger
  • Copy the two finalfiltered.csv file generated above and the idsex file over.
  • Change the TNOP in Rscript Manual_UKB_FinalStretch.R to sample size.
  • Run: Rscript MergerOfFinalDF.R
  • FinalFiltered.tsv is the final output to be used in STEP3.

STEP2_MCHIP

chmod +x *

cp ../STEP1/mchip_sample/mutect2.mchip.filter.1.vcf ./
tail -n +32 mutect2.mchip.filter.1.vcf > mutect2.mchip.filternh.1.vcf
mv mutect2.mchip.filternh.1.vcf Input.vcf
(echo -e "X.CHROM\tPOS\tID\tREF\tALT\tQUAL\tFILTER\tINFO\tFORMAT\tSAMPLE"; cat Input.vcf) > Input.vcf
  • Run: . ./Manual_START_UKB_Filter.sh
  • Then: Rscript Manual_UKB_FinalStretch.R

Now copy the MCHIP and LCHIP FinalFiltered.csv file to STEP3, along with the idsex.txt file.

STEP3

Run Final_summary.R and generate files for MCHIP and LCHIP