Feasibility study of a single‐primer extension‐based microhaplotype NGS system

DNA degradation has been a thorny problem in forensic science. Shortening the amplicon length of the genetic markers improves the analysis of degraded DNA effectively. Microhaplotype (MH) has been proposed as a potential genetic marker that can be used for degraded DNA analysis. In the present study, a 146-plex MH-next-generation sequencing (NGS) system with an average Ae of 6.876 was constructed. Unlike other MH studies, a single-primer extension (SPE)-based NGS library preparation method was used to improve the detection of MH markers for degraded DNA. SPE employs a locus-specific and universal primer to amplify target fragments, reducing the necessity for complete fragment sequences. SPE might effectively mitigate the impact of degradation on amplification. However, SPE produces amplicons of varying lengths, posing challenges in allele calling for SPE-NGS data. To address this issue, this study proposed a flexible allele-calling strategy to improve amplicon detection. In addition, this study evaluated the forensic efficacy of the system using 12 low-template samples (from 1 ng to 7.8 pg), 10 mock-degraded DNA with various degrees of degradation, and 8 forensic casework samples. When the template is as low as 7.8 pg, our system can accurately detect at least 37 loci and achieves a random match probability (RMP) of 10^-30 using the complete allele-calling strategy. Eighty-two loci can be detected, and RMP can reach 10^-54 using a flexible allele-calling strategy. After 150 min of 98°C treatment, 36 loci can still be detected, and an RMP of 10^-5 can be obtained using the flexible allele-calling strategy. Furthermore, the number of single nucleotide polymorphism detected at different DNA amounts and degradation levels suggests that the SPE method combined with a flexible allele-calling strategy is effective.