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GATK4.0.9.0 undetected a delins site

qiuqiu87qiuqiu87 Member
Dear GATK team,
I use GATK4.0.9.0 Haplotypercaller to detect germline variation, and find a delins site is not call. As the figure show, this is gvcf output, 32944606 is delTTT, and 32944609 is insAAAA. But the vcf output only is delTTT, insAAAA is filtered. And I find the in GATK4.1.1.0 and 4.1.2.0 also not call insAAAA in vcf. The site is verified by Sanger, and is a delinsAAAA. So why GATK4.0.9.0 filtered the insAAAA in 32944609 ?

The vcf of GATK4.0.9.0:
chr13 32944606 . CTTT C 9318.60 . AC=1;AF=0.500;AN=2;BaseQRankSum=1.294;DP=814;ExcessHet=3.0103;FS=1.093;MLEAC=1;MLEAF=0.500;MQ=60.03;MQRankSum=0.000;QD=11.90;ReadPosRankSum=0.290;SOR=0.769 GT:AD:DP:GQ:PL 0/1:423,360:783:99:9326,0,45236
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Answers

  • bshifawbshifaw admin Member, Broadie, Moderator admin
    edited June 2

    Hi @qiuqiu87,

    "HaplotypeCaller performs a local reassembly and realignment of the reads in the region surrounding potential variant sites" then the tool calls the variants. If you would like to know more about how haplotypecaller calls variants you can read through Local re-assembly and haplotype determination.
    The site in question may not have met the default threshold to be considered a variant by Haplotypecaller due to quality of evidence. However, the tool does have parameters that can be tweaked to be more sensitive to calling variants which can be found in the tool documentation page (warning, this increases the chance of false positives).

    If you believe the tool is misbehaving please read the following doc Generate a "bamout file" showing how HaplotypeCaller has remapped sequence reads to generate a bamout and a IGV screenshot of the region not being called and post it here.

    Post edited by bshifaw on
  • qiuqiu87qiuqiu87 Member
    Hi @bshifaw
    The screenshot showed the raw bam and bamout about the not being called region. The two bam files have reads support the variant.
  • bshifawbshifaw admin Member, Broadie, Moderator admin

    I'll ask the dev team, in the meantime tell us more about the background. e.g. What's its origin (human, exome), what is the exact command you used, what was the sequencing platform, are you working with amplicon data?

  • qiuqiu87qiuqiu87 Member
    @bshifaw
    Thanks for your answer. The command I used is the default command with -O to output vcf. The data is about human sequence, the tumor detection panel with amplicon data. And as I paste the gvcf output, the qual of 32944609 is 0, so is filtered before output vcf.
  • qiuqiu87qiuqiu87 Member
    @bshifaw
    And I try some versions, and find 4.0.9.0 and higher version can't detect the insAAAA variants.
  • bshifawbshifaw admin Member, Broadie, Moderator admin

    I've asked the dev team, I'll let you know what they say.
    In the meantime let me know if using --adaptive-pruning and --kmer-size 35 as suggested in this forum post helps
    https://gatkforums.broadinstitute.org/gatk/discussion/comment/57464#Comment_57464

  • qiuqiu87qiuqiu87 Member
    Thanks, I tried both to use --adaptive-pruning true and to change the length of kmer-size 35, the variant in 32944609 is still missing in vcf. But I found in the gvcf output, the quality of 32944609 from 0 increase to 2451.05, but still filtered in vcf output.
  • bshifawbshifaw admin Member, Broadie, Moderator admin

    Then from this point the above message may apply

    The site in question may not have met the default threshold to be considered a variant by Haplotypecaller due to quality of evidence. However, the tool does have parameters that can be tweaked to be more sensitive to calling variants which can be found in the tool documentation page (warning, this increases the chance of false positives).

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