Development and validation of a novel 29-plex Y-STR typing system for forensic application

Short tandem repeat within the male-specific part of the human Y chromosome (Y-STR) is an effective forensic tool in mixture identification, patrilineal relationship evaluation, and familial searches. Despite their usefulness, current Y-STR-based genotyping systems often lack the discriminatory power to resolve genetic relationships between distant relatives or within patrilocal populations. In this study, we developed a novel Y-STR 29-plex typing system, which combined the 17 Y-STR loci used in the AmpFLSTR® Yfiler® PCR Amplification Kit (Yfiler), eight Y-STR loci with a low-medium mutation rate, and four rapidly mutating Y-STR loci. The system was generated to achieve greater discriminatory power between male subjects and improved ability to infer haplogroup classifications. The system was extensively tested on data from 752 individuals for its sensitivity, male specificity, species specificity, mixture resolution, reproducibility, concordance, stutter and size accuracy, precision, and population genetics, following the Scientific Working Group on DNA Analysis Methods (SWGDAM) guidelines. The results demonstrated that the Y-STR 29-plex typing system was time-efficient, reproducible, accurate, sensitive, and robust to familial searching and paternal biogeographic ancestry inference.