Selection of microhaplotype loci and development of panel for forensic application

Abstract Microhaplotypes have gained significant attention in forensic genetics research due to their advantageous characteristics, including low mutation rates, absence of stutter products, short fragment lengths, and high polymorphism. These features position them as promising tools for various forensic applications. In this study, using the Illumina NovaSeq 6000 platform, a 29‐plex microhaplotype (MH) panel was developed for the Chinese Han population. The panel's forensic utility was systematically evaluated across multiple applications, including paternity testing, individual identification, population analysis, and mixture analysis. In a cohort of 444 Chinese Han individuals, the panel exhibited robust performance: the average effective number of alleles (A e ) was 3.2938, and the total discrimination power (TDP) and the cumulative power of exclusion (CPE) reached 0.9999999999999999999999926 and 0.9999998228, respectively, demonstrating its high informativeness for individual identification and paternity testing. Simulated pairing analysis showed comparable efficacy to traditional STR systems in paternity testing. Excluding six microhaplotypes not included in 1000 Genomes, the remaining 23 microhaplotypes can effectively distinguish the five populations and can be used for population analysis. The observed allele coverage ratios closely matched expected values in artificial mixtures tested, indicating that the panel could play a complementary role in mixture analysis. A consistency rate of 98.5% was observed in paired normal/tumor samples, indicating potential for assisting tumor origin identification. In conclusion, the developed 29‐plex microhaplotype panel offers significant value for forensic applications, including individual identification, paternity testing, population analysis, and supplementary roles in mixture/tumor sample analysis. This study establishes a novel and effective tool for forensic genetic practice.