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GVCF of amplicon sequencing


I'm trying to generate a GVCF with HaplotypeCaller based on amplicon (PCR) sequencing, and bases towards the end of the reads (~10 bases last bases) consistently get genotype quality of 0, even when the base quality is high. I tried simulating reads, and even if the position is covered by reads from both strands, with a large number of reads, and high base quality the genotype quality of the reference is 0. If there is a SNP in that position, it does get called with reasonable genotype quality, only the reference bases get quality 0. I tried changing various parameters but nothing seems to make a difference? Is there a parameter I'm missing? should I submit a detailed bug report?


  • bhanuGandhambhanuGandham Cambridge MAMember, Administrator, Broadie, Moderator admin
    edited August 14

    Hi @ulitskyi

    1) What version of gatk are you using.
    2)Please post the exact command you are using
    3) Have you taken a look at the bamout for the regions with GQ = 0?
    4) Please post records of the variants with GQ = 0

    Post edited by bhanuGandham on
  • ulitskyiulitskyi Member ✭✭

    1) I'm using
    2) java -Xmx6g -jar ../gatk- HaplotypeCaller -R ~/gatk/hg19.unmasked.fa -G StandardAnnotation -mbq 17 --standard-min-confidence-threshold-for-calling 25 --max-reads-per-alignment-start 0 --disable-read-filter NotDuplicateReadFilter --min-dangling-branch-length 5 --allow-non-unique-kmers-in-ref --kmer-size 30 --kmer-size 10 --kmer-size 15 -ERC GVCF -I read1.sampe.bam -O small.i.vcf -L small.intervals --recover-all-dangling-branches --assembly-region-out test.txt --dont-trim-active-regions --min-assembly-region-size 28

    3) The bamout does seem to include the region of interest where GT=0 (see image https://us.v-cdn.net/5019796/uploads/editor/ye/ty6e3xa8vqom.png "")

    4) this it the relevant region in the VCF:
    chr1 14155328 . G . . END=14155401 GT:DP:GQ:MIN_DP:PL 0/0:10:30:10:0,30,385
    chr1 14155402 . G A, 414.02 . DP=10;ExcessHet=3.0103;MLEAC=2,0;MLEAF=1.00,0.00;RAW_MQandDP=36000,10 GT:AD:DP:GQ:PL:SB 1/1:0,10,0:10:30:428,30,0,428,30,428:0,0,10,0
    chr1 14155403 . T . . END=14155440 GT:DP:GQ:MIN_DP:PL 0/0:10:30:10:0,30,426
    chr1 14155441 . G . . END=14155596 GT:DP:GQ:MIN_DP:PL 0/0:0:0:0:0,0,0

    This is a region if I'm altering the read to include a SNP in the region where there is a GT=0:
    chr1 14155328 . G . . END=14155401 GT:DP:GQ:MIN_DP:PL 0/0:100:99:100:0,120,1800
    chr1 14155402 . G A, 4486.03 . DP=100;ExcessHet=3.0103;MLEAC=2,0;MLEAF=1.00,0.00;RAW_MQandDP=360000,100 GT:AD:DP:GQ:PL:SB 1/1:0,100,0:100:99:4500,301,0,4500,301,4500:0,0,100,0
    chr1 14155403 . T . . END=14155445 GT:DP:GQ:MIN_DP:PL 0/0:100:99:100:0,120,1800
    chr1 14155446 . C . . END=14155446 GT:DP:GQ:MIN_DP:PL 0/0:100:0:100:0,0,0
    chr1 14155447 . T G, 1829.60 . BaseQRankSum=0.000;DP=100;ExcessHet=3.0103;MLEAC=1,0;MLEAF=0.500,0.00;MQRankSum=0.000;RAW_MQandDP=360000,100;ReadPosRankSum=0.00GT:AD:DP:GQ:PL:SB 0/1:50,50,0:100:99:1837,0,1837,1988,1988,3976:50,0,50,0
    chr1 14155448 . T . . END=14155596 GT:DP:GQ:MIN_DP:PL 0/0:0:0:0:0,0,0

    Issue · Github
    by bhanuGandham

    Issue Number
    Last Updated
  • bhanuGandhambhanuGandham Cambridge MAMember, Administrator, Broadie, Moderator admin

    Hi @ulitskyi

    I am sorry I meant GQ, genotype quality = 0

  • ulitskyiulitskyi Member ✭✭

    That's ok I meant GQ as well :) - you can see that chr1 14155446 and chr1 14155448-chr1 14155455 get GQ=0 here, even though they are all covered by hiqh quality reads, both in the input BAM and in the bamout. Please advise...

  • bhanuGandhambhanuGandham Cambridge MAMember, Administrator, Broadie, Moderator admin

    Can you please send me your gvcf and bamout using instructions provided here: https://software.broadinstitute.org/gatk/guide/article?id=1894

  • ulitskyiulitskyi Member ✭✭

    uploaded ulitsky.zip - please let me know if you see it

  • bhanuGandhambhanuGandham Cambridge MAMember, Administrator, Broadie, Moderator admin


    I am looking into this and will get back to you shortly.

  • bhanuGandhambhanuGandham Cambridge MAMember, Administrator, Broadie, Moderator admin

    Hi @ulitskyi

    This might be a bug in the code specific to amplicon sequencing. I have opened a github issue ticket for it and you can track its progress here: https://github.com/broadinstitute/gatk/issues/6100

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