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ECNT Value in Mutect2

Hello,

I am using Mutect2 and FilterMutectCalls to call variants in mtDNA. According to the vcf file, the value recorded for ECNT is "Number of events in this haplotype". I am assuming that this is the number of times that a particular mutation was found in all the reads under that base pair. I am concerned because in my data, I am seeing clusters of mutations with the same ECNT value. For example:

#CHROM POS ID REF ALT QUAL FILTER INFO FORMAT
gi|9626243|ref|NC_001416.1| 115 . C T VL PASS DP=445;ECNT=6;POP_AF=5.000e-08;P_CONTAM=0.00;P_GERMLINE=-7.025e+01;TLOD=6.86 GT:AD:AF:F1R2:F2R1:MBQ:MFRL:MMQ:MPOS:SA_MAP_AF:SA_POST_PROB
gi|9626243|ref|NC_001416.1| 157 . T A VL PASS DP=427;ECNT=6;POP_AF=5.000e-08;P_CONTAM=0.00;P_GERMLINE=-1.113e+02;TLOD=8.55 GT:AD:AF:F1R2:F2R1:MBQ:MFRL:MMQ:MPOS:SA_MAP_AF:SA_POST_PROB
gi|9626243|ref|NC_001416.1| 470 . C T VL PASS DP=703;ECNT=9;POP_AF=5.000e-08;P_CONTAM=0.00;P_GERMLINE=-1.859e+02;TLOD=15.58 GT:AD:AF:F1R2:F2R1:MBQ:MFRL:MMQ:MPOS:SA_MAP_AF:SA_POST_PROB
gi|9626243|ref|NC_001416.1| 500 . A T VL PASS DP=691;ECNT=9;POP_AF=5.000e-08;P_CONTAM=0.00;P_GERMLINE=-1.927e+02;TLOD=7.59 GT:AD:AF:F1R2:F2R1:MBQ:MFRL:MMQ:MPOS:SA_MAP_AF:SA_POST_PROB
gi|9626243|ref|NC_001416.1| 601 . CT C VL PASS DP=671;ECNT=9;POP_AF=5.000e-08;P_CONTAM=0.00;P_GERMLINE=-1.766e+02;RPA=3,2;RU=T;STR;TLOD=7.68 GT:AD:AF:F1R2:F2R1:MBQ:MFRL:MMQ:MPOS:SA_MAP_AF:SA_POST_PROB
gi|9626243|ref|NC_001416.1| 635 . C T VL PASS DP=665;ECNT=9;POP_AF=5.000e-08;P_CONTAM=0.00;P_GERMLINE=-1.602e+02;TLOD=34.37 GT:AD:AF:F1R2:F2R1:MBQ:MFRL:MMQ:MPOS:SA_MAP_AF:SA_POST_PROB
gi|9626243|ref|NC_001416.1| 645 . C T VL PASS DP=668;ECNT=9;POP_AF=5.000e-08;P_CONTAM=0.00;P_GERMLINE=-1.818e+02;TLOD=7.87 GT:AD:AF:F1R2:F2R1:MBQ:MFRL:MMQ:MPOS:SA_MAP_AF:SA_POST_PROB
gi|9626243|ref|NC_001416.1| 704 . T TAAAAAA VL PASS DP=660;ECNT=9;POP_AF=5.000e-08;P_CONTAM=0.00;P_GERMLINE=-1.845e+02;RPA=5,11;RU=A;STR;TLOD=5.89 GT:AD:AF:F1R2:F2R1:MBQ:MFRL:MMQ:MPOS:PGT:PID:SA_MAP_AF:SA_POST_PROB
gi|9626243|ref|NC_001416.1| 736 . A G VL PASS DP=666;ECNT=6;POP_AF=5.000e-08;P_CONTAM=0.00;P_GERMLINE=-1.723e+02;TLOD=20.03 GT:AD:AF:F1R2:F2R1:MBQ:MFRL:MMQ:MPOS:SA_MAP_AF:SA_POST_PROB
gi|9626243|ref|NC_001416.1| 754 . A G VL PASS DP=654;ECNT=6;POP_AF=5.000e-08;P_CONTAM=0.00;P_GERMLINE=-1.606e+02;TLOD=29.79 GT:AD:AF:F1R2:F2R1:MBQ:MFRL:MMQ:MPOS:SA_MAP_AF:SA_POST_PROB
gi|9626243|ref|NC_001416.1| 788 . A T VL PASS DP=639;ECNT=6;POP_AF=5.000e-08;P_CONTAM=0.00;P_GERMLINE=-1.738e+02;TLOD=6.65 GT:AD:AF:F1R2:F2R1:MBQ:MFRL:MMQ:MPOS:SA_MAP_AF:SA_POST_PROB
gi|9626243|ref|NC_001416.1| 898 . C T VL PASS DP=671;ECNT=6;POP_AF=5.000e-08;P_CONTAM=0.00;P_GERMLINE=-1.821e+02;TLOD=5.38 GT:AD:AF:F1R2:F2R1:MBQ:MFRL:MMQ:MPOS:SA_MAP_AF:SA_POST_PROB
gi|9626243|ref|NC_001416.1| 958 . A G VL PASS DP=671;ECNT=6;POP_AF=5.000e-08;P_CONTAM=0.00;P_GERMLINE=-1.847e+02;TLOD=6.69

Is there an explanation for why the data would look like this? It seems odd that the mutations would be occurring exactly the same number of times as the variants surrounding it.

Thank you,

kzwon

Answers

  • bhanuGandhambhanuGandham Cambridge MAMember, Administrator, Broadie, Moderator admin

    Hi @kzwon

    ECNT is the number of potential (pre-filtering) somatic mutations (events) exhibited in an assembled haplotype. That is, if a mutation is found on a locally-assembled haplotype with two other mutations, each one gets an ECNT of 3. It has nothing to do with individual reads except insofar as haplotypes are assembled from reads.
    Does this help answer your question?

  • Brunods10Brunods10 AugustaMember
    Hello @bhanuGandham.

    I'm trying to understand the FilterMutectCalls filters and I don't understand the "haplotype" filter. I have read the description in my VCF file: "Variant near filtered variant on same haplotype.", and I've read the definition in the Mutect2 pdf:
    """
    J. Bad Haplotypes
    Mutect2 emits phasing information for calls in the same assembly region. We assign a “bad haplotype” probability equal to the greatest technical artifact probability of any in-phas call within a certain distance, by default 100 bases.
    """

    I have also read about the threshold "ECNT" which is defined as "Number of events in this haplotype".

    Your answer above helped me understand how the ECNT value is determined, but why is it a bad thing to have an ECNT value greater than 1? Why don't the variants get a PASS?
  • akovalskakovalsk Member, Broadie, Moderator admin

    @Brunods10

    Would you mind sharing the pdf where it says the ECNT score is causing the variant to be filtered?

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