Mitochondrial Calling
Typically, there are approximately 100 mitochondria in each mammalian cell. Each mitochondrion harbors 2–10 copies of mitochondrial DNA (mtDNA). For example, if 20% of the chrM copies have a variant, then the allele frequency (AF) is 20%. This is also referred to as continuous allele frequency. The expectation is that the AF of variants on chrM is anywhere between 0% and 100%.
DRAGEN processes chrM through a continuous AF pipeline
QUAL is not output in the chrM variant records. Instead, the confidence score is FORMAT/SQ, which gives the Phred-scaled confidence that a variant is present at a given locus.
##FORMAT=<ID=SQ,Number=A,Type=Float,Description="Somatic quality">
GQ is not output in the chrM variant records because DRAGEN doesn't test for multiple diploid genotype candidates. Instead, an ALT allele is considered as a candidate variant. If FORMAT/SQ> vc-sq-call-threshold (default = 3.0), then the FORMAT/GT is set to 0/1. The FORMAT/AF yields an estimate on the variant allele frequency, which ranges anywhere within [0,1]. For very high allele frequencies (AF ≥ 95%) the FORMAT/GT is set to 1/1.
You can apply the following filters to mitochondrial variant calls.
• | --vc-sq-call-threshold |
Set the SQ threshold for emitting calls in the VCF. The default is 0.1.
• | --vc-sq-filter-threshold |
Set the SQ threshold to mark emitted VCF calls as filtered. The default is 3.0.
• | --vc-enable-triallelic-filter |
Enable the multiallelic filter. The default value is false.
• | If FORMAT/SQ < vc-sq-call-threshold, the variant is not output in the VCF. |
• | If FORMAT/SQ > vc-sq-call-threshold but FORMAT/SQ < vc-sq-filter-threshold the variant is output in the VCF but FILTER=weak_evidence. |
• | If FORMAT/SQ> vc-sq-call-threshold, FORMAT/SQ > vc-sq-filter-threshold, and no other filters are triggered, the variant is output in the VCF and FILTER=PASS. |
The following are example VCF records on the chrM. The examples shows one call with very high AF and another with very low AF. In both cases FORMAT/SQ > vc-sq-call-threshold. FORMAT/SQ is also > vc-sq-filter-threshold, so the FILTER annotation is PASS.
chrM 513 . GCA G . PASS DP=4937;MQ=235.28;FractionInformativeReads=0.883
GT:SQ:AD:AF:F1R2:F2R1:DP:SB:MB 1/1:95.46:33,4327:0.992:7,1081:26,3246:4360:31,2,2371,1956:10,23,2811,1516
chrM 1713 . A G . PASS DP=7175;MQ=165.91;FractionInformativeReads=0.995
GT:SQ:AD:AF:F1R2:F2R1:DP:SB:MB 0/1:21.49:7122,20:0.003:3066,10:4056,10:7142:3896,3226,8,12:3605,3517,10,10

In NON_REF regions, the FORMAT/GT is hard-coded to 0/0 and at variant loci, the FORMAT/GT is hard-coded to 0/1.
The FORMAT/GT for chrM is not determined by FORMAT/AF. The FORMAT/GT is determined by whether a variant is emitted at a position or not. The decision to emit the variant or not is determined by comparing the FORMAT/SQ score to a threshold. All variants with FORMAT/SQ > vc-sq-call-threshold get emitted. Variants with FORMAT/SQ < vc-sq-call-threshold are not emitted in the gVCF and are banded in a NON_REF region with FORMAT/GT=0/0.
One of the following two scenarios can occur at a given position.
• | A variant is detected by the genotyper at a given position and the FORMAT/SQ > vc-sq-call-threshold. In this case, the FORMAT/GT is hard-coded to 0/1. DRAGEN outputs FORMAT/AD, FORMAT/DP, and FORMAT/AF computed at that position. |
• | No variant is detected at the given position, or a variant is detected but the FORMAT/SQ < vc-sq-call-threshold. In this case, the FORMAT/GT is hard-coded to 0/0. The positions are banded with contiguous positions if the positions are in the same scenario. All positions with FORMAT/GT = 0/0 are banded together. The FORMAT/DP reported for the band is computed as the median DP across all positions within the band. The FORMAT/AD values reported in the band are the AD values selected at the position where FORMAT/DP = median DP. In the joint VCF, FORMAT/AF is computed based on FORMAT/AD. |
The following is an example of a variant record on chrM in a trio joint VCF. The variant was detected in the second sample with a confidence score that passed the filter threshold. In the first and third samples GT=0/0, which indicates a tentative hom-ref call (ie, that position for the sample is in a NON_REF region over which no variant was detected with sufficient confidence), but the weak_evidence filter tag indicates that this call is made with low confidence.
chrM 2623 . A G . . DP=18772;MQ=111.77 GT:AD:AF:DP:FT:SQ:F1R2:F2R1 0/0:6841,7:0.001:4334:weak_evidence:0:.:. 0/1:6736,2053:0.234:8789:PASS:21.32:3394,1060:3342,993 0/0:6086,9:0.001:5613:weak_evidence:0:.:.