Development and Evaluation of a Novel Microhaplotype Panel for DNA Mixture and Kinship Analysis

Microhaplotypes (MHs), together with the development of next-generation sequencing (NGS), hold great promise for forensic genetics, particularly for mixture interpretation and kinship analysis. In this study, we performed genome-wide screening for new MH markers composed of two or more variants (InDels or single nucleotide polymorphism [SNP]) within 300 bp, based on the Chinese Southern Han (CHS) population from the 1000 Genomes Project (1KG). We developed a 31-plex MH panel specifically for the Chinese Han population, consisting of six MHs and 25 compound markers. The panel demonstrated high polymorphism, with an average effective number of alleles (A_e) of 5.10. The combined power of discrimination (CPD) and cumulative probability of exclusion (CPE) reached 1-1.58 × 10^-29 and 1-1.24 × 10^-13, respectively. Subsequently, allele detection rates and unique allele detection rates of minor contributors were evaluated in two- to four-person DNA mixtures with varying input levels and contributor ratios. The panel maintained stable detection performance under low-template or moderately unbalanced conditions, with significant declines observed only when both factors were present. The panel also demonstrated great capacity for close relatives (first-degree and second-degree relatives). Parent-child and full-sibling pairs were completely distinguished from unrelated individuals, with consistently strong classification performance in both simulated datasets and real pedigrees. The mean log₁₀ likelihood ratio (LR) values for second-degree relative pairs provided moderate to strong evidential support, whereas discrimination of third- to fifth-degree relative pairs remained limited. In conclusion, these findings indicate that the panel is a valuable supplementary tool for forensic DNA analysis, particularly for mixture deconvolution and kinship inference.