Massively parallel sequencing of 74 microhaplotypes for relationship testing in U.S. populations

Abstract

Microhaplotypes (MHs) are increasingly recognized as valuable forensic markers due to their low mutation rates, lack of stutter artifacts, and compatibility with massively parallel sequencing (MPS). These biomarkers have shown utility in human identification, mixture deconvolution, ancestry prediction and kinship testing. Although familial relationship testing has historically relied on fragment-sized analysis of STRs, it is now evolving with the advent of both MPS and MHs. While single nucleotide polymorphism (SNP) microarrays have also been used for kinship testing, they require high-quality and high-quantity DNA and are ineffective in the presence of mixtures—limitations that MHs can overcome due to their short amplicons and ability to deconvolute mixed DNA samples. This study specifically aimed to evaluate the performance of a previously designed 74-locus Ion AmpliSeq™ MH panel on the Ion GeneStudio S5 platform and compare it against a 29-fragment sized STR panel for kinship testing in four major U.S. populations. The 74plex MH panel was sequenced using a sample set of 372 family samples across African American, European American, Southwest Hispanic, and East Asian American population groups. Its performance was assessed on Familias software in differentiating between close and distant relatives, which included parent-child, full siblings, half-siblings, and cousins. For parent-child relationships, MHs and STRs showed clear separation, but MHs yielded consistently higher log₁₀LR values (>20) with tighter clustering, suggesting stronger discriminatory power than STRs. With full siblings, MHs consistently outperformed STRs, with their distributions showing higher log₁₀LRs. However, for half-siblings, both genetic markers struggled with significant overlap between related and unrelated pairs, though MHs showed higher log₁₀LR values than STRs. Neither 74 MHs nor 29 STRs were able to distinguish between cousins, exhibiting extensive overlap and log₁₀LRs near or below zero. In summary, the 74-locus Ion AmpliSeq™ MH panel offers enhanced discriminatory power for close kinship, particularly first-degree relationships. While MHs enhance second-degree resolution, the 74-MH struggles with more distant relationships like cousins. These findings underscore the need for larger MH panels (≥200 loci) to reliably resolve distant kinship, supporting their broader integration into forensic applications including kinship analysis, mixture deconvolution, and ancestry inference.