= 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 ==

GitHub link: https://github.com/molgenis/ngs-utils/blob/master/scripts/vcf-fill-gtc.pl
Raw version for wget: https://raw.githubusercontent.com/molgenis/ngs-utils/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
}}}