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P079 – Enhancing the Analysis of Highly Degraded DNA: A Novel Strategy Combining ddPCR for Quality Assessment and a Short-Amplicon Multi-InDel Panel for Genotyping
Abstract
Forensic analysis of highly degraded biological evidence, such as ancient bones, charred remains, or aged stains, remains a significant challenge because of extensive DNA fragmentation, frequently causing conventional STR kits to fail. To address these limitations, this study proposes a novel workflow integrating droplet digital PCR (ddPCR) for precise quantification and degradation assessment, followed by genotyping with a short-amplicon Multi-InDel panel. Unlike traditional qPCR, ddPCR provides absolute quantification without standard curves. We utilized a triplex ddPCR system capable of simultaneously detecting three DNA fragments of different lengths (75 bp, 145 bp, and 235 bp) to determine absolute copy numbers and degradation indices. On the basis of these quality metrics, a customized Multi-InDel panel targeting 26 loci with amplicons strictly controlled under 125 base pairs was employed for identification. The workflow was rigorously evaluated using artificially degraded DNA, actual casework samples, and historically inconclusive specimens. The results demonstrated that triplex ddPCR accurately assessed DNA degradation levels, optimizing downstream input. Furthermore, compared with conventional STRs, the short-amplicon Multi-InDel panel significantly improved allele detection rates and full profile recovery, particularly when standard amplification yielded partial or no data. Statistical analysis confirmed that the panel maintained high discriminatory power even with compromised templates. In conclusion, the synergy of ddPCR-based quality assessment and short-amplicon Multi-InDel genotyping offers a robust, sensitive solution for challenging forensic casework. This strategy provides reliable results from highly degraded materials where traditional methods fall short, ultimately aiding in the resolution of cold cases and disaster victim identification.
Keywords
Highly Degraded DNA, ddPCR, Multi-InDel, Forensic Genetics
Authors
- Jienan Li (Xiangya School of Basic Medical Science, Central South University, China)
- Xiaoyi Fu (Xiangya School of Basic Medical Science, Central South University, China)
- Lian Liu (Xiangya School of Basic Medical Science, Central South University, China)
- Xinxin Du (Xiangya School of Basic Medical Science, Central South University, China)
- Yilin Shen (Xiangya School of Basic Medical Science, Central South University, China)
- Qiheng Xie (Xiangya School of Basic Medical Science, Central South University, China)
- Feifei zheng (Xiangya School of Basic Medical Science, Central South University, China)
- Yuepeng Wang (Xiangya School of Basic Medical Science, Central South University, China)
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