Y-chromosomal short tandem repeats (Y-STRs) serve as highly informative tools in forensic genetics, particularly for tracing paternal lineages and generating investigative leads when autosomal STR profiling is uninformative. Nevertheless, the discriminating power of expanded Y-STR panels and the optimal strategies for database searching remain insufficiently defined. In this study, we first introduce kinshipY, an interactive online platform designed to streamline the analysis of Y-STR mutations, visualization of pedigree structures, calculation of genetic distances, and evaluation of statistical power. Using this tool, we empirically estimated the mutation rates of 80 Y-STRs included in the Forensic Analysis System Multiplecues SetB Kit based on 488 father–son pairs. We further evaluated the differentiation rates among male relatives and between unrelated males using three deep-rooted families spanning 1–27 meioses. A total of 120 mutations were identified, yielding an overall mutation rate of 3.1 × 10⁻³ (95% CI: 2.5 × 10⁻³–3.7 × 10⁻³). Single-step mutations accounted for 95% of events, with gains and losses occurring at comparable frequencies. The SetB panel distinguished 27.96% of father–son pairs and 50% of siblings, achieving 100% differentiation for relationships separated by ≥11 meioses. To differentiate males from distinct lineages, we established a thresholding strategy that balanced the false positive rate (FPR) and false negative rate (FNR), demonstrating that step-difference-based thresholds outperformed locus-difference-based thresholds. For the SetB panel, a step-difference threshold of ≥15 enabled differentiation of 99.59% of unrelated males. In contrast, panels with fewer Y-STRs—Yfiler (16 Y-STRs), Class A (19 Y-STRs), Yfiler Plus (25 Y-STRs), and Class A+B (32 Y-STRs)—exhibited significantly higher FPR and FNR. Collectively, this study highlights the enhanced resolution afforded by the SetB panel. For Y-STR database searching, we recommend: (1) using high-resolution marker sets whenever possible; (2) adopting a step-difference-based matching strategy; (3) applying dynamic, panel-specific thresholds; and (4) incorporating more distant relatives for threshold estimation when integrating forensic investigative genetic genealogy. These recommendations may provide valuable guidance for forensic practice.

Authors

• Xiaoyang Ma (Sun Yat-sen University, China)
• Ran Li (Sun Yat-sen University, China)
• Hongyu Sun (Sun Yat-sen University, China)