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, which is similar to the somatic variant calling pipeline. In this case, a single ALT allele is considered and the AF is estimated. The estimated AF can be anywhere between 0% and 100%. The default variant AF thresholds are applied to mitochondrial variant calling.

• --vc-enable-af-filter-mito

Whether to enable the allele frequency for mitochondrial variant calling. The default is true.

• --vc-af-call-threshold-mito

Set the threshold for emitting calls in the VCF. The default is 0.01.

• --vc-af-filter-threshold-mito

Set the threshold to mark emitted vcf call as filtered. The default is 0.02.

QUAL and GQ are 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. A call is made if FORMAT/SQ>vc-sq-call-threshold (default = 3.0).

##FORMAT=<ID=SQ,Number=A,Type=Float,Description="Somatic quality">

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 show one call with very high AF and another with 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 7028 . C T . PASS DP=8868;MQ=60.19;FractionInformativeReads=0.993

GT:SQ:AD:AF:F1R2:F2R1:DP:SB:MB 0/1:21.48:8622,181:0.021:4190,82:4432,99:8803:4344,4278,94,87:5032,3590,101,80

For homref calls (eg in NON_REF regions of gVCF output) the FORMAT/GT is hard-coded to 0/0. The FORMAT/AF yields an estimate on the variant allele frequency, which ranges anywhere within [0,1]. For variant calls with FORMAT/AF < 95%, the FORMAT/GT is set to 0/1. For variants with very high allele frequencies (FORMAT/AF ≥ 95%), the FORMAT/GT is set to 1/1.

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 . PASS 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:.:.