A bioinformatic method to predict the 5' ends of primer sequences in multiple STR kits

Short tandem repeat (STR) kits, which enable the detection of genetic variations, similarities, and origins, are essential for forensic analysis. However, human-specific primers vary depending on the manufacturer, resulting in mismatched profiles, consequently limiting the kit efficiency. Efficient methods for acquiring primer information to elucidate the reasons behind the allelic discrepancies are yet to be developed. This study aimed to develop an analytical method for directly reading labeled PCR fragments using an Illumina next-generation sequencer. In this study, DNA libraries from seven commercial STR kits (GlobalFiler, GF; Yfiler Plus, YFP; AmpFLSTR Identifiler Plus, IDP; AmpFLSTR MiniFiler, MF; and AmpFLSTR Yfiler, YF; as well as the PowerPlex Fusion, PPF and PowerPlex 16 System, PP16) were sequenced into pair-end reads, and bioinformatic analysis was performed to predict the 5' ends of the primer sequences in each kit. The 5' ends of the primer sequences corresponding to peaks in the evaluated STR kits were further compared with published sequences to validate positional accuracy. The 5' ends of primer pairs at all loci predicted in this study were perfectly matched to those of published primer pairs in the PP16 kits. Our method elucidated the reconstruction process from fluorescent peaks in the electropherogram into a sequence-based histogram for forensic massively parallel sequencing analysis. Moreover, the effect of single nucleotide polymorphism-specific primers in the GF was revealed. Our findings demonstrate that this method can be used to accurately identify the cause of mismatched profiles between kits or low amplification at a specific locus.