Forensic evaluation of 101 identity-informative SNPs in a Filipino population using massively parallel sequencing

DNA-based human identification traditionally relies on short tandem repeats (STRs) as the primary genetic markers for analysis. However, single nucleotide polymorphisms (SNPs), particularly identity-informative SNPs (iiSNPs) located on autosomes, offer distinct advantages for analyzing degraded DNA because of their low mutation rates and short amplicon lengths. Massively parallel sequencing (MPS) has enhanced forensic capabilities by enabling the simultaneous analysis of large numbers of STRs and SNPs in a single assay. This study characterized 101 iiSNPs in a representative Filipino population using two commercially available MPS kits (Precision ID and ForenSeq™ DNA Signature Prep Kits), contributing data from an underrepresented Southeast Asian population to forensic DNA databases. Genotype data from 143 unrelated Filipino males from the National Capital Region (NCR) and surrounding areas were analyzed to estimate allele frequencies, forensic efficiency parameters, Hardy-Weinberg equilibrium (HWE), linkage disequilibrium (LD), population structure, and kinship performance for robust statistical interpretation in casework. All markers conformed to HWE expectations after correction, and no significant LD was detected among iiSNPs or between iiSNPs and 23 autosomal STRs (aSTRs) previously generated using the PowerPlex® 21 System and Verifiler™ Direct PCR Amplification Kit. This supports the assumption of marker independence and the application of standard forensic statistical models. Of the 101 iiSNPs evaluated, 64 met the criteria for broad forensic applicability. Both iiSNP panels demonstrated strong individualization power, with combined match probabilities five- to six-fold lower than those of 23 aSTRs. Population genetic analyses indicated that Filipinos cluster closely with East Asian populations relative to the other populations included in the analyses, while exhibiting distinct genetic characteristics. This underscores the importance of population-specific reference data. Furthermore, simulated kinship analyses showed that iiSNPs performed comparably to aSTRs for first-degree relationships and provided added value when combined with aSTRs for second-degree relationships. In contrast, neither the iiSNP-only, aSTR-only, nor the combined aSTR-iiSNP marker systems were sufficient to reliably resolve third-degree relationships, highlighting the need for higher-density iiSNP panels. This study established the first Filipino forensic iiSNP database and validated the forensic applicability of 101 iiSNPs included in two commercial MPS panels. Future studies should extend the analysis to other Philippine population groups, including Indigenous cultural communities (ICCs) and Indigenous peoples (IPs), which remain culturally and geographically distinct from mainstream populations. Additionally, refining universal iiSNP panels and exploring higher-density MPS datasets may help identify new markers to further strengthen forensic DNA profiling in criminal investigations in the Philippines.