Evaluation of the MHSeqTyper47 kit for forensically challenging DNA genotyping

Abstract

Microhaplotypes have been highly regarded for forensic mixture DNA deconvolution because they do not experience interference from stutters in the same way as short tandem repeat markers, and they tend to be more polymorphic than single nucleotide polymorphism markers. However, forensic microhaplotype kits have not been reported. The MHSeqTyper47 kit genotypes 47 microhaplotype loci, with A_e (effective number of alleles) values of 29 of these loci higher than 3.00. In this study, MiSeq FGx sequencing metrics for MHSeqTyper47 were presented, and the genotyping accuracy of this kit was examined. The sensitivity of MHSeqTyper47 reached 62.5pg, and full genotyping results were obtained from degraded DNA samples with degradation indexes ≤ 3.00. Full genotypes were obtained in the presence of 100ng/μL tannin, 50μM heme, 25ng/μL humic acid, and 1.25μg/μL indigo dye. In DNA mixture studies, a minimum of 31 loci of the minor contributor were correctly genotyped at 1:99 or 99:1 mixing ratios, with the cumulative random matching probability of these loci reaching 4.54 × 10^-25. Mixing ratios could be reliably predicted from two-donor DNA mixtures based on the loci with four called alleles. Taken together, these data showed that the MHSeqTyper47 kit was effective for forensically challenging DNA analysis.