A comprehensive evaluation of mutation rates and male differentiation using an 80-Y-STR panel.

Y-chromosomal short tandem repeats (Y-STRs) are highly informative tools in forensic investigations for tracing paternal lineages and generating investigative leads when direct autosomal STR matches are not available. However, the discriminative power of expanded Y-STR panels and optimal strategies for database searching remain inadequately defined. Here, we first presented kinshipY, an interactive online platform that streamlines the analysis of Y-STR mutations, pedigree structure visualization, genetic distance calculation, and statistical power evaluation. Using this tool, we then empirically estimated the mutation rates of 80 Y-STRs (Forensic Analysis System Multiplecues SetB Kit) based on 488 father-son pairs. Finally, the differentiation rates among male relatives and between unrelated males were evaluated using three deep-rooted families spanning 1-27 meioses. The results showed that a total of 120 mutations were identified, yielding an overall mutation rate of 3.1 × 10⁻³ (95 % CIs: 2.5 × 10-3 - 3.7 × 10-3). Single-step mutations accounted for 95 % of events, with gains and losses occurring at nearly equal frequencies. The SetB panel distinguished 27.96 % of father-son pairs and 50 % of siblings, with differentiation rates reaching 100 % for relationships separated by ≥ 11 meioses. To differentiate males from distinct lineages, we established a threshold‑setting strategy that balances both the false positive rate (FPR) and false negative rate (FNR), demonstrating that step‑difference‑based thresholds outperform locus‑difference‑based thresholds. For the SetB panel, a step‑difference threshold of ≥ 15 enabled the 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. In summary, this study demonstrates the enhanced resolution offered by the SetB panel. For Y-STR database searching, we recommend the following: (1) use a powerful marker set whenever possible; (2) adopt a step-difference-based matching strategy; (3) apply dynamic, panel-specific thresholds; and (4) when integrating forensic investigative genetic genealogy, include more distant relatives for threshold estimation. These recommendations could provide valuable guidance for forensic practice.