Running the VCF aggregration and de-sampling procedure

1. Install VCFtools and Tabix

Download and install from: ​http://sourceforge.net/projects/samtools/files/tabix/ and ​http://vcftools.sourceforge.net/ Tabix needs to be compiled first. (use MAKE )

2. Add location of VCFtools and Tabix to your path

E.g. use

export PATH=/Volumes/Users/Software?/vcftools_0.1.10/bin/:/Volumes/Users/Software?/tabix-0.2.6/:${PATH}

Or a more permanent option (.bashrc file or so)

3. Download and install Perl if you don't have it

Go to: ​https://www.perl.org/

4. Download the script and put in folder of choosing

​https://github.com/molgenis/ngs-utils/blob/master/scripts/vcf-fill-gtc.pl

Aggregation procedure

1. Merge sample VCFs into one batch VCF

vcf-merge CAR_*/*.vcf.sorted.filtered.gz | bgzip -c > merged.vcf.gz

2. Create a summary VCF per batch

vcf-fill-gtc.pl -vcfi merged.vcf.gz -vcfo stripped.vcf -ss -fv PASS -si -ll INFO > stripped.vcf.log

The option -ss is crucial here: it removed all sample details.

Afterwards, be sure to inspect the log file for warnings!

more stripped.vcf.log

Full manual:

Create a summary VCF per batch:

-ss : remove sample details! -fv PASS : keep only high quality variant calls that pass all filters applied in NextGene?.

Just to be sure: variants should already have been filtered on PASS only in a previous step, so this should be redundant here...

-si : remove all INFO subfields except for INFO:AN and INFO:AC.

INFO:AN and INFO:AC were automatically updated by vcf-merge, but the others were not and may contain erroneous annotation that cause vcf-validator to complain the created VCF is not valid.

-ll : specifies log level, e.g. INFO

Troubleshooting

Q: My VCF files are not completely valid format! A: The are some built-in options to help with this. For example:

Prepare sample VCFs for one batch; e.g. CAR_Batch1_106Samples cd /Volumes/CardioKitVCFs/OriginalVCFs/CAR_Batch1_106Samples Fix missing '>' at the end of contig meta-data lines.

perl -pi -e 's/(contig=<ID=[^{>\n]+)$/$1>/' CAR_*/*.vcf}

Sort, filter on 'PASS', bgzip and index with tabix (vcftools will not work on uncompressed, unindexed VCF files.)

for item in $(ls CAR_*/*.vcf); \ do echo "Processing $item..."; \ vcf-sort $item | vcf-annotate -H > $item\.sorted\.filtered; \ bgzip $item\.sorted\.filtered; \ tabix -p vcf $item\.sorted\.filtered\.gz; \ done