Pairwise kinship testing with microhaplotypes: Can advancements be made in kinship inference with these markers?

Kinship testing based on genetic relatedness is one of the major tasks in forensic genetics. Although short tandem repeats (STRs) are the "gold standard" biomarkers for relationship testing, microhaplotypes (MHs) have also emerged as viable options for kinship elucidation. In this work, the kinship testing efficiency of 54 highly polymorphic MHs was studied in two extended families consisting of parent-offspring, full siblings, grandparent-grandchildren, uncle/aunt-nephew/nieces, and first cousins. In addition, ten-thousand pairs of different degrees of relationships were simulated using various datasets including 54 MHs, 27 STRs plus 94 single nucleotide polymorphisms (SNPs) that were included in the ForenSeq DNA Signature Prep Kit (ForenSeq), 54 MHs plus loci in ForenSeq, and different subsets of 417-published MHs. The panels' system effectiveness in the kinship analysis were accessed by likelihood ratio distributions. The results showed that 54 MHs could be used in first-degree relationship testing with high reliability. The effectiveness of 54 MHs was slightly lower than ForenSeq but only by a narrow margin. Both 54 MHs and ForenSeq were not sufficient for distant relationship testing, and approximately 200 microhaplotypes with an average expected heterozygosity (He) = 0.79 were enough to determine second-degree relationships, but a panel of 417 MHs with an average He = 0.72 was not sufficient to first cousins testing according to the simulation analysis. In conclusion, 54 MHs could be used to serve as supplement markers for kinship testing; and well-established STR markers plus well-performing microhaplotype markers may become collective tools in forensic applications, though an enlarged pool of forensic markers is needed for distant relationship testing.