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What should I use as known variants/sites for running tool X?

edited January 2016 in FAQs

1. Notes on known sites

Why are they important?

Each tool uses known sites differently, but what is common to all is that they use them to help distinguish true variants from false positives, which is very important to how these tools work. If you don't provide known sites, the statistical analysis of the data will be skewed, which can dramatically affect the sensitivity and reliability of the results.

In the variant calling pipeline, the only tools that do not strictly require known sites are UnifiedGenotyper and HaplotypeCaller.

Human genomes

If you're working on human genomes, you're in luck. We provide sets of known sites in the human genome as part of our resource bundle, and we can give you specific Best Practices recommendations on which sets to use for each tool in the variant calling pipeline. See the next section for details.

Non-human genomes

If you're working on genomes of other organisms, things may be a little harder -- but don't panic, we'll try to help as much as we can. We've started a community discussion in the forum on What are the standard resources for non-human genomes? in which we hope people with non-human genomics experience will share their knowledge.

And if it turns out that there is as yet no suitable set of known sites for your organisms, here's how to make your own for the purposes of BaseRecalibration: First, do an initial round of SNP calling on your original, unrecalibrated data. Then take the SNPs that you have the highest confidence in and use that set as the database of known SNPs by feeding it as a VCF file to the base quality score recalibrator. Finally, do a real round of SNP calling with the recalibrated data. These steps could be repeated several times until convergence. Good luck!

Some experimentation will be required to figure out the best way to find the highest confidence SNPs for use here. Perhaps one could call variants with several different calling algorithms and take the set intersection. Or perhaps one could do a very strict round of filtering and take only those variants which pass the test.

2. Recommended sets of known sites per tool

Summary table

Tool dbSNP 129 dbSNP >132 Mills indels 1KG indels HapMap Omni
RealignerTargetCreator X X
IndelRealigner X X
BaseRecalibrator X X X
(UnifiedGenotyper/ HaplotypeCaller) X
VariantRecalibrator X X X X
VariantEval X

RealignerTargetCreator and IndelRealigner

These tools require known indels passed with the -known argument to function properly. We use both the following files:

• Mills_and_1000G_gold_standard.indels.b37.sites.vcf

• 1000G_phase1.indels.b37.vcf (currently from the 1000 Genomes Phase I indel calls)

BaseRecalibrator

This tool requires known SNPs and indels passed with the -knownSites argument to function properly. We use all the following files:

• The most recent dbSNP release (build ID > 132)

• Mills_and_1000G_gold_standard.indels.b37.sites.vcf

• 1000G_phase1.indels.b37.vcf (currently from the 1000 Genomes Phase I indel calls)

UnifiedGenotyper / HaplotypeCaller

These tools do NOT require known sites, but if SNPs are provided with the -dbsnp argument they will use them for variant annotation. We use this file:

• The most recent dbSNP release (build ID > 132)

VariantRecalibrator

For VariantRecalibrator, please see the FAQ article on VQSR training sets and arguments.

VariantEval

This tool requires known SNPs passed with the -dbsnp argument to function properly. We use the following file:

• A version of dbSNP subsetted to only sites discovered in or before dbSNP BuildID 129, which excludes the impact of the 1000 Genomes project and is useful for evaluation of dbSNP rate and Ti/Tv values at novel sites.
Post edited by Geraldine_VdAuwera on

Geraldine Van der Auwera, PhD

Tagged:

Questions and comments up to August 2014 have been moved to an archival thread here:

Geraldine Van der Auwera, PhD

• United StatesMember Posts: 2

When you say to use "both" files for known indels, how do you parse this in the command?

Is the following command correct?

GATK.sh \
-T RealignerTargetCreator \

Yes that's correct, @traversc.

Geraldine Van der Auwera, PhD

• United StatesMember Posts: 2

Thank you!

• GreensboroMember Posts: 71

@Geraldine_VdAuwera said:
Questions and comments up to August 2014 have been moved to an archival thread here:

The link for "What are the standard resources for non-human genomes?" is not working. Let me know if there is any updated link

Geraldine Van der Auwera, PhD

• Member Posts: 46

At least for BQSR and IndelRealigner purposes, should I use the "all" or "common" dbSNP dataset?

@johnma
Hi,

I'm not sure I understand your question. What do you mean by all and common?

Thanks,
Sheila

• Member Posts: 46

"Common" is a subset of "All" where in at least one population the MAF is larger than or equal to 0.01. See http://www.ncbi.nlm.nih.gov/variation/docs/human_variation_vcf/.

@johnma
Hi,

I don't think it will really make a large difference. In Indel Realignment, you will just be testing realignment around more positions if you use "all". In Base Recalibration, it is better to overmask sites a little bit than to undermask them.

-Sheila

• Member Posts: 6

Hi,
For RealignerTargetCreator and IndelRealigner, why is 1000G_phase1.indels recommended instead of phase3? Isn't phase3 more updated? And why Mills_and_1000G_gold_standard.indels is chosen over dbSNP?
Thanks,
Wen