Interpreting alt_allele_in_normal filter in Mutect2

darioberdariober Cambridge UKMember

Hi- If I understand correctly Mutect2, the alt_allele_in_normal flag is assigned when variant reads in the normal exceed the --max_alt_alleles_in_normal_count setting (which has default=1).

I wonder if this generates false negatives since you can have only few variant reads in normal out of many total reads. For example, in normal you can have REF:ALT=1000:3 and in tumour REF:ALT=1000:700. These numbers would pass the NLOD and TLOD filters (right?) but the variant would be flagged as alt_allele_in_normal. So I wonder whether one should rescue alt_allele_in_normal variants by, for example, applying a simple fisher test to see if the ALT counts are convincingly larger in tumour vs normal.

Just to make a concrete example, this is a variant that potentially may be rescued. Here tumour REF:ALT=35:25 and normal REF:ALT=51:2:

chr17 39524885 . G A . alt_allele_in_normal ECNT=1;HCNT=1;MAX_ED=.;MIN_ED=.;NLOD=6.35;TLOD=76.43 GT:AD:AF:ALT_F1R2:ALT_F2R1:FOXOG:QSS:REF_F1R2:REF_F2R1 0/1:35,25:0.396:7:18:0.28:1386,995:12:23 0/0:51,2:0.039:2:0:1:2028,82:26:25

(This comment is based on Mutect2 in GATK 3.8.0, I would switch to GATK4 but I see Mutect is still in beta there)

Many thanks
Dario

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