Characterizing the amplification of STR markers in multiplex polymerase chain displacement reaction using massively parallel sequencing

Polymerase chain displacement reaction (PCDR) showed advantages in forensic low-template DNA analysis with improved amplification efficiency, higher allele detection capacity, and lower stutter artifact than PCR. However, characteristics of STR markers after PCDR amplification remain unclarified for the limited resolving power of capillary electrophoresis (CE). This issue can be addressed by massively parallel sequencing (MPS) technology with higher throughput and discriminability. Here, we developed a multiplex PCDR system including 24 STRs and amelogenin. In addition, a PCR reference was established for comparison. After amplification, products were subjected to PCR-free library construction and sequenced on the Illumina NovaSeq system. We implemented a sequence-matching pipeline to separate different amplicon types of PCDR products from the combination of primers. In the sensitivity test, the PCDR multiplex obtained full STR profiles with as low as 125 pg 2800M control DNA. Based on that, single-source DNA samples were tested. First, highly concordant genotypes were observed among the PCDR multiplex, the PCR reference, and CE-based STR kits. Next, read counts of different PCDR amplicon types were investigated, showing a relative abundance of 78:12:12:1 for the shortest amplicon S, the two medium amplicons M1 and M2, and the longest amplicon L. We also analyzed the stutter artifacts for distinct amplicon types, and the results revealed the reduction of N - 1 and N - 2 contraction stutters, and the increase of N + 1 and N + 2 elongation stutters in PCDR samples. Moreover, we confirmed the feasibility of PCDR for amplifying degraded DNA samples and unbalanced DNA mixtures. Compared to the previous proof of principle study, our work took a further step to characterize the complete profile of STR markers in the PCDR context. Our results suggested that the PCDR-MPS workflow is an effective approach for forensic STR analysis. Corresponding findings in this study may help the development of PCDR-based assays and probabilistic methods in future studies.