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

Geraldine_VdAuweraGeraldine_VdAuwera Cambridge, MAMember, Administrator, Broadie admin
edited April 2018 in Frequently Asked Questions

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.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.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 Sheila on

Comments

  • Geraldine_VdAuweraGeraldine_VdAuwera Cambridge, MAMember, Administrator, Broadie admin

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

    http://gatkforums.broadinstitute.org/discussion/4555/questions-about-known-variant-resources

  • traversctraversc United StatesMember

    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 \
    -R /data2/broad_refs/b37/human_g1k_v37.fasta \
    -o /data2/broad_refs/b37/human_g1k_v37_indel.intervals \
    -known /data2/broad_refs/b37/Mills_and_1000G_gold_standard.indels.b37.vcf \
    -known /data2/broad_refs/b37/1000G_phase1.indels.b37.vcf

  • Geraldine_VdAuweraGeraldine_VdAuwera Cambridge, MAMember, Administrator, Broadie admin

    Yes that's correct, @traversc.

  • traversctraversc United StatesMember
  • everestial007everestial007 GreensboroMember ✭✭

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

    http://gatkforums.broadinstitute.org/discussion/4555/questions-about-known-variant-resources

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

  • Geraldine_VdAuweraGeraldine_VdAuwera Cambridge, MAMember, Administrator, Broadie admin
  • johnmajohnma Member

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

  • SheilaSheila Broad InstituteMember, Broadie ✭✭✭✭✭

    @johnma
    Hi,

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

    Thanks,
    Sheila

  • johnmajohnma Member

    "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/.

  • SheilaSheila Broad InstituteMember, Broadie ✭✭✭✭✭

    @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

  • wenluowenluo Member

    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

  • shleeshlee CambridgeMember, Broadie ✭✭✭✭✭

    Hi Wen (@wenluo),

    The 1KG phase1 set represents more common variant sites whereas the phase3 set includes many more sites, including sites variant in the population at low frequency. The population VCF is meant to ensure that these common sites are part of the targeted regions in case your BAM inputs do not trigger these sites as targets. Adding in the additional sites provided by phase3 data provides incremental value while adding a whole lot of compute.

    If I recall correctly, the Mills indels are preferable because they were validated using orthogonal approaches. DbSNP is known to contain many artifacts as anyone can submit a variant.

  • mcvumcvu IrelandMember

    Regarding:
    "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)"

    • does this mean all simultaneously, or any of the following?

    Having looked at what's available in the bundle to download, there are two files:
    Mills_and_1000G_gold_standard.indels.b37.vcf.gz
    Mills_and_1000G_gold_standard.indels.hg19.sites.vcf.gz

    But there is no "Mills_and_1000G_gold_standard.indels.b37.sites.vcf" that I can see. Which of the above two would be most appropriate to use?

  • shleeshlee CambridgeMember, Broadie ✭✭✭✭✭

    Hi @mcvu,

    You can see on this page how we apply the resources in VQSR.

    The b37 and hg19 references are different and so you should use the one that matches your alignments.

  • jpfloridojpflorido SevilleMember

    According to best practices (GATK 3.8) , bases should be recalibrated before variant calling. However, not sure which -knownSites files should be used given the fact I have used GRch37p13 reference genome for mapping. The only one I'm sure is dbSNP file, since I have downloaded the latest one (150) related to the aforementioned reference genome from NCBI.
    However, having a look into GATK's resource bundle, I can find Mills_and_1000G_gold_standard.indels.{ref}.b37.sites.vcf and 1000G_phase1.indels.{ref}.vcf where ref={b37, hg19}. Which one should I use?

    Many thanks,
    Javier

  • SheilaSheila Broad InstituteMember, Broadie ✭✭✭✭✭

    @jpflorido
    Hi Javier,

    I think you should use the b37 version. Have a look at this thread as well.

    -Sheila

  • jpfloridojpflorido SevilleMember

    @Sheila
    Thanks! The only thing is that I had to change from chr? identifiers to just ? (for example, from chr1 to 1) in my reference genome (GRch37p13) since 1000G_phase1.indels.b37.vcf and Mills_and_1000G_gold_standard.indels.b37.vcf files do not have 'chr' in the chromosomes ids.

  • SheilaSheila Broad InstituteMember, Broadie ✭✭✭✭✭

    @jpflorido
    Hi,

    You should not have to change anything in your contigs if you use the correct reference. Can you check if hg19 files work with your VCF?

    Thanks,
    Sheila

  • jpfloridojpflorido SevilleMember

    @Sheila
    Thanks for you reply! The thing is that I've downloaded GRCh37.p13 from https://www.gencodegenes.org/releases/19.html . This reference has chr prefix in contig names (couldn't find GRCh37.p13 at https://console.cloud.google.com/storage/browser/genomics-public-data/references/ ).
    That's the reason I had to rename contigs in my reference genome .
    Not sure whether using hg19 vcf versions of 1000G and Mills were appropriated, since my reference genome was GRCh37.p13, not hg19...
    Thanks again!
    Javier

  • SheilaSheila Broad InstituteMember, Broadie ✭✭✭✭✭

    @jpflorido
    Hi Javier,

    So, your reference and BAM files all have chr in front of the contig name? The hg19 reference also has chr in front of the contig names.

    If the b37 files and hg19 files we have are not compatible with your data, I am afraid we cannot help. You will have to do some research and find files compatible with the reference you are using.

    -Sheila

  • jpfloridojpflorido SevilleMember

    @Sheila,
    No problem, hg19 reference genome downloaded from UCSC did the trick.
    Thanks for your help!
    Javier

  • meysam_roodimeysam_roodi TorontoMember

    Hi,
    It is recommended on GATK website that indelrealigner step could safely be ignored if haplotype caller is to be used for variant calling. So, how the indelrealigner specific known sites are to be used?

  • SheilaSheila Broad InstituteMember, Broadie ✭✭✭✭✭

    @meysam_roodi
    Hi,

    This is an older article. We leave it up to help people who still choose to run the Indel Realignment steps. You do not have to run it, as it is no longer Best Practice. Have a look at this blog post.

    -Sheila

  • kchang3kchang3 Member
    edited June 2018

    I don't see 1000G_phase1.indels in hg38 gatk-bundle. Is it something being prepared for? At the mean time, is the best way to just lift over the hg19 1000G phase indels coordinates to hg38?

    edit: I found out Homo_sapiens_assembly38.known_indels.vcf.gz can be used to replace 1000G phase 1 indels according to Sheila on this thread https://gatkforums.broadinstitute.org/gatk/discussion/1213/whats-in-the-resource-bundle-and-how-can-i-get-it

    Post edited by kchang3 on
  • TamaraDTamaraD Member

    Hi,
    I am working on human target exome data and I am trying to follow the GATK Best Practice Germline SNPs and Indels workflow. Also, I will be using the hg38 as a reference genome.

    Can I use the latest version of dbsnp 151 for the Base Recalibrator and Haplotype Caller? Is this choice according to GATK Best Practice?

    Thanks,
    Tamara

  • TamaraDTamaraD Member

    Also, for Base Recalibrator I would use Mills_and_1000G_gold_standard.indels.hg38.vcf and 1000G_phase1.snps.high_confidence.hg38.vcf and for
    Variant Recalibrator: Mills_and_1000G_gold_standard.indels.hg38.vcf
    hapmap_3.3.hg38.vcf. and
    1000G_omni2.5.hg38.vcf. All from the bundle/hg38. Is it suitable for target exome data Germline SNPs and Indels variant calling?

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  • syer89syer89 Member

    Hi, wanted to confirm if the recommendation of using known sites from all 3 sources - dbsnp, Mills and 1KG indels still applicable for GATK4 BQSR step? Thanks!

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