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

Area of Science:

• Forensic genetics
• Population genetics
• Next-generation sequencing technologies

Background:

• Microhaplotypes (MHs) are emerging forensic markers with advantages over traditional short tandem repeats (STRs) for human identification.
• MHs offer benefits like low mutation rates, no stutter, and compatibility with massively parallel sequencing (MPS), enabling applications in kinship testing and mixture deconvolution.
• Existing kinship testing methods using SNP microarrays have limitations with DNA quality, quantity, and mixtures, which MHs can potentially overcome.

Purpose of the Study:

• To evaluate the performance of a 74-locus microhaplotype (MH) panel on the Ion GeneStudio S5 platform for kinship testing.
• To compare the discriminatory power of the 74-locus MH panel against a 29-locus STR panel across four major U.S. populations.
• To assess the utility of MHs in differentiating various degrees of familial relationships, including parent-child, full siblings, half-siblings, and cousins.

Main Methods:

• Sequencing of a 74-plex MH panel using 372 family samples from African American, European American, Southwest Hispanic, and East Asian American populations.
• Performance assessment using Familias software to differentiate related and unrelated individuals.
• Comparison of log₁₀LR values generated by MHs and STRs for different relationship types.

Main Results:

• The 74-locus MH panel demonstrated superior performance over the 29-locus STR panel for parent-child and full-sibling relationships, yielding higher log₁₀LR values.
• Both MHs and STRs showed limitations in distinguishing half-siblings due to overlap between related and unrelated pairs, although MHs provided higher log₁₀LRs.
• Neither the 74-locus MH panel nor the 29-locus STR panel could reliably differentiate cousins, showing extensive overlap in log₁₀LR values.

Conclusions:

• The 74-locus Ion AmpliSeq™ MH panel enhances discriminatory power for close kinship (first-degree relatives) and improves resolution for second-degree relatives.
• Larger microhaplotype panels (≥200 loci) are necessary for reliable identification of distant familial relationships.
• These findings support the broader integration of MHs into forensic science for kinship analysis, mixture deconvolution, and ancestry inference.