Annotating Variants: Introduction
As we've already seen, determining the presence or absence of a variant from NGS data is not trivial. It is software dependent and has inherent trade-offs between sensitivity and specificity. Inevitably, the number of putative variants in a real data set is very large; for example the first samples from the 1000 Genomes project typically found 0.1% variants (3 million variants), approximately 10% of which had never been previously observed (300,000 novel variants per individual.) False positive discovery rates are also typically very high at this stage.
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Because these tools draw in information from may disparate sources, they can be very difficult to install, configure, use, and maintain. For example, the vcf
files from the 1000 Genomes project are arranged in a deep ftp tree by date of data generation. Large genome centers spend significant resources managing these tools.
Pre-packaged programs
Annovar - one of the most powerful yet simple to run variant annotators available
Annovar is a variant annotator. Given a vcf file from an unknown sample and a host of existing data about genes, other known SNPs, gene variants, etc., Annovar will place the discovered variants in context.
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This next exercise will give you some idea of how Annovar works; we've taken the liberty of writing the bash script annovar_pipe.sh
around the existing summarize_annovar.pl
wrapper (a wrapper within a wrapper - a common trick) to even further simplify the process for this course.
Exercise:
First, look at the code for our annovar_pipe.sh
command.
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cp $BI/ngs_course/human_variation/*chrom20.GATK* . |
ANNOVAR output
Annovar does a ton of work in assessing variants for us (though if you were going for clinical interpretation, you still have a long way to go - compare this to RUNES or CarpeNovo). It provides all these output files:
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Everything after the "LJB_GERP++" field in exome_summary came from the original VCF file, so this file REALLY contains everything you need to go on to functional analysis! This is one of the many reasons I like Annovar.
Scavenger hunts!
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The final answer is "DEFB126"
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The final form of the command that you had was:grep "frameshift" NA12878.chrom20.GATK.vcf.exome_summary.csv | awk -F"," '{print $2"\t"$3}' | uniq -c | sort -r
Some questions for thought:
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Other variant annotators:
- http://www.yandell-lab.org/software/vaast.html
- http://www.broadinstitute.org/gatk/gatkdocs/ VariantAnnotator annotations
- http://www.bioconductor.org/help/workflows/variants/
- http://vat.gersteinlab.org/
- http://code.google.com/p/mu2a/
Notes
Variants consist of single base base changes, insertions and deletions, and larger scale structural changes. "Larger scale" is usually defined relative to the capabilities of the technology; for example, a "small indel" usually means "detectable within a single sequence read". In 2009, sequence reads were about 50 bp but in 2011 they were 100 bp.