A massively parallel sequencing assay of microhaplotypes for mixture deconvolution

Abstract Microhaplotypes (microhaps or MHs) are markers characterized by a set of single nucleotide polymorphisms (SNPs) within an expanse of 300 bp exhibiting multiple allelic combinations. Microhap alleles within a locus have all the same size, do not generate stutter artifacts, and have low mutation rates. These features complement short tandem repeats (STRs) for human identification and mixture deconvolution. The analysis of microhaps is enhanced by massively parallel sequencing (MPS) that enables the determination of the parental SNP haplotypes by clonal sequencing of each strand. This study aims to evaluate the mixture performance of a 74 microhap locus-assay on the Ion S5TM and compare the results to size-based and sequence-based STR analysis. The detection limit of the panel was tested and the panel’s performance evaluated in parallel with GlobalFilerTM kit and Precision ID GlobalFilerTM NGS STR Panel v2. An assay of two-to-five person mixtures was simulated at different contribution ratios and amounts of DNA (1 ng and 10 ng) to mimic casework-like samples. The 74-locus panel was sensitive down to 50 pg input DNA. Size-based STR mixtures analysis was challenging, but it could be improved by MPS when the sequence of minor alleles was different from the stutter sequence. For two-person mixtures, full microhap profile of the minor donor could be reported at 1:10 ratio with minimal allele/locus dropout at 20:1, and more significant at higher mixture ratios. For three-to-five person mixtures, full microhap profiles were reported for all minor donors with the exception of some loci that underperformed at imbalanced mixture ratios. These findings suggest that microhaps can improve mixture deconvolution and supplement STR typing analysis.