Development and validation of a multiplex panel with 232 microhaplotypes and software for forensic kinship analysis

In this study, we developed and validated a novel microhaplotype (MH) panel, the FGID Microhaplotype Kit, which contains 232 loci and was specifically designed for forensic kinship analysis. The performance of the panel was evaluated through rigorous testing that included sensitivity, species specificity, inhibitor resistance, uniformity, stability, accuracy and mixture deconvolution. The results showed that the kit is capable of reliably detecting all loci with minimal DNA input. It showed high species specificity for 12 non-human DNA samples and resistance to common inhibitors. In addition, forensic statistical analysis revealed a combined discriminatory power (cDP) of 1-1.68e-223 and superior combined exclusion power for duo and trio cases compared to standard STR panels. The panel was also tested for kinship analyzes with simulated and real pedigree samples and showed significantly higher likelihood ratios (LR) for detecting relationships between parents and offspring, full siblings, half siblings and first cousins, especially for more distant kinship types where conventional STR panels have difficulties. Using the FGID kinship software with the MH panel significantly improved the accuracy of kinship analysis, allowing even closely related individuals to be effectively discriminated while reducing the number of false negatives. In addition, principal component analysis (PCA) showed that the panel can distinguish the major world populations and East Asian subpopulations. Taken together, these results suggest that the FGID Microhaplotype Kit and associated software provide an efficient and accurate solution for forensic kinship analysis that offers better discriminatory power and reliability than traditional STR-based methods.