Developmental validation of a six-dye Multi-InDels kit for personal identification and paternity testing of highly degraded DNA samples.

Abstract

Highly degraded samples are common in forensic casework and remain among the most challenging samples for forensic genetic analysis. Conventional STR-based technologies often yield partial or null profiles from such samples, making the acquisition of complete profiles a persistent challenge. To address this limitation, we developed a six-dye Multi-InDels kit based on Multi-InDels markers characterized by short amplicon sizes, high polymorphism, and low mutation and recombination rates, while maintaining full compatibility with capillary electrophoresis platforms. The kit integrates 26 Multi-InDels loci and an amelogenin locus into a multiplex system, with all amplicons not exceeding 125 bp, and is specifically designed for personal identification and paternity testing involving highly degraded DNA. Comprehensive internal validation studies demonstrated robust system performance, with complete profiles consistently obtained from as little as 62.5 pg of input DNA. Comparative analyses with STR kits showed that the Multi-InDels kit markedly improved the recovery of complete profiles from highly degraded samples, including artificially degraded DNA and formalin-fixed paraffin-embedded tissue. Population studies yielded a combined random match probability (CMP) of 2.25 × 10⁻¹⁷ and a cumulative probability of exclusion (CPE) of 0.999925. In paternity testing, 48 confirmed trios produced an average combined paternity index (CPI) of 3.2952 × 10⁶, and all 4,800 non-biological fathers were successfully excluded. In addition, innovative investigations were conducted on Multi-InDels allele nomenclature, evaluation strategies for degraded samples, and rare alleles. Collectively, these findings demonstrate that the Multi-InDels kit provides a convenient, feasible, and robust tool for forensic analysis of highly degraded samples.