The degradation index (DI), obtained from quantitative PCR, is widely used in forensic DNA analysis to assess DNA degradation prior to STR and Y-STR profiling. However, DI primarily reflects DNA fragmentation and does not directly account for other forms of DNA damage, raising questions about its ability to represent functional DNA integrity across different degradation mechanisms. In this study, we investigated the relationship between DI and STR/Y-STR profiling performance using multiple degradation models, including enzymatic fragmentation, ultraviolet (UV) irradiation, and heat-induced degradation. Human genomic DNA samples were subjected to controlled degradation using these models, followed by quantitative PCR using the Quantifiler HP DNA Quantification Kit to obtain DI values. STR and Y-STR analyses were performed using the GlobalFiler and Yfiler Plus kits across a range of DNA input amounts. Our results demonstrated that the relationship between DI and profiling performance is strongly dependent on the degradation mechanism. In enzymatically fragmented DNA, allele detection rates and peak heights showed predictable relationships with DI values, and nearly complete profiles were obtained at DI values up to approximately 2.0 when sufficient DNA input was used. UV-irradiated DNA exhibited similar trends, although amplification efficiency decreased more gradually with increasing DI, consistent with damage mechanisms dominated by base modifications such as photoproducts rather than extensive strand breakage. In contrast, heat-induced degradation showed a distinct pattern, particularly under low-template conditions. Substantial reductions in amplification efficiency were observed even at relatively low DI values (≈1.1–1.2), indicating that factors beyond fragmentation contribute to reduced PCR performance. At higher DI values, a biphasic pattern was observed in low-molecular-weight loci, and the Y-STR locus DYS385 exhibited a unique non-monotonic amplification pattern, indicating locus-dependent responses to degradation. These findings suggest that heat-induced DNA damage involves not only strand fragmentation but also chemical base modifications that impair polymerase activity. Collectively, these results demonstrate that DI has intrinsic limitations as a surrogate for functional DNA integrity, as it does not capture all types of DNA damage. While DI can assist in estimating template input when the degradation mechanism is known, its interpretation must be contextualized with respect to the underlying damage processes. Overall, our findings highlight the importance of integrating degradation mechanisms with DI-based assessment to improve the reliability of STR and Y-STR interpretation, particularly in challenging contexts such as thermally degraded and low-template samples.

Authors

• Seiki Nakao (Osaka Medical and Pharmaceutical University, Japan)
• Jun Yoshida (Osaka Medical and Pharmaceutical University, Japan)
• Kazuya Mori (Osaka Medical and Pharmaceutical University, Japan)
• Misa Kitagawa (Osaka Medical and Pharmaceutical University, Japan)
• Takako Sato (Osaka Medical and Pharmaceutical University, Japan)