= Running the VCF aggregration and de-sampling procedure =

== 1. Install VCFtools and Tabix ==

Download and install [http://sourceforge.net/projects/samtools/files/tabix/ Tabix] and [http://sourceforge.net/projects/vcftools/files/ VCFtools]. Tabix needs to be compiled first, so use ''make''.

== 2. Add location of VCFtools and Tabix to your path ==

E.g. use

{{{
export PATH=/Applications/vcftools_0.1.10/bin/:/Applications/tabix-0.2.6/:$PATH
}}}

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

Also, add the VCFtools Perl library to the Perl5 library locations:

{{{
export PERL5LIB=/Applications/vcftools_0.1.11/perl/:$PERL5LIB
}}}

== 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:

{{{
wget https://raw.githubusercontent.com/molgenis/ngs-utils/master/scripts/vcf-fill-gtc.pl --no-check-certificate
}}}

= Aggregation procedure =

== 1. Sort, filter, bgzip and index ==

VCFtools will not work on uncompressed, unindexed VCF files so we must sort, filter on 'PASS', bgzip and index with tabix.

{{{
for item in $(ls mydirectory/*.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
}}}


== 2. Merge sample VCFs into one batch VCF ==

{{{
vcf-merge mydirectory/*.vcf.sorted.filtered.gz | bgzip -c > merged.vcf.gz
}}}

== 3. Create a summary VCF per batch ==

{{{
perl 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
}}}




== Troubleshooting ==

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

Fix missing '>' at the end of contig meta-data lines.

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

Q: I get this error:

{{{
Can't locate Log/Log4perl.pm in @INC
}}}

A: Install Log4perl:

{{{
cpan Log::Log4perl
}}}

Q: What are the script options?
A: Man page:

{{{
#
# 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.
#
}}}