Application of a short amplicon triallelic SNP panel for parentage analysis of highly degraded DNA

Genetic typing of highly degraded DNA remains one of the most critical challenges in forensic genetics, particularly in parentage analysis, where relationship assessment is needed despite limited genetic information. To address this, we developed and validated a high efficiency multiplex system comprising 25 triallelic single nucleotide polymorphisms (SNPs) specifically optimized for highly degraded DNA, with all internal amplicons shorter than 110 bp. Validation following SWGDAM guidelines demonstrated a sensitivity threshold of 0.0625 for 100% allele detection rate (ADR), with consistent genotypes obtained down to 0.03125 ng. Performance stability was confirmed through the successful genotyping of artificially degraded DNA (up to 60 min) and formalin fixed paraffin embedded (FFPE) samples (74% average allelic call rate). Population genetic analysis of 230 unrelated individuals from Changsha, China, showed no deviation from Hardy Weinberg equilibrium (HWE), providing an outstanding cumulative matching probability (CMP) of 1.59 × 10^-18 and a cumulative power of exclusion (CPE) of 0.999979. In a validation study of 91 trios, the panel showed 100% concordance with STR based results, achieving a mean cumulative paternity index (CPI) of 5.9 × 10⁶ and resolved potential miscalls caused by a rare primer binding site mutation at rs3857817 through the implementation of degenerate primers. This triallelic SNP panel offers a high performance, cost effective tool for parentage analysis in challenging forensic scenarios involving highly degraded biological evidence.