Development and validation of a 365-Plex NGS panel for integrated Y-STR and Y-SNP typing: Enhancing paternal lineage...

In forensic genetics, Y-chromosomal short tandem repeats (Y-STRs) and single nucleotide polymorphisms (Y-SNPs) are indispensable for paternal lineage identification and population genetic research. While Y-STRs enable short-term familial linkage, their high mutation rates complicate precise lineage determinations. Conversely, Y-SNPs provide stable deep phylogenetic resolution, complementing Y-STRs by resolving mutation-induced ambiguities and enhancing forensic accuracy. However, existing methods lack a combined detection system tailored to Chinese populations that integrates Y-STRs (with varied mutation rates) and Y-SNPs optimized for diverse forensic scenarios. To address this gap, we developed a 365-plex next-generation sequencing (NGS) panel optimized for Chinese populations. The panel integrates 57 Y-STRs (13 rapidly mutating, 44 moderate-to-slow) and 308 Y-SNPs (68 % targeting O subclades), with short amplicons designed for degraded DNA. Validation following SWGDAM guidelines confirmed its reliability. It has a genotyping sensitivity of 125 pg input DNA, 100 % repeatability, and can resolve male-male mixtures up to 1:4 and male-female mixtures up to 1:10 and no cross-reactivity in non-target samples. Among 326 Shandong Han Chinese males, the panel achieved perfect Y-STR haplotype diversity (HD=1.0000) and discrimination capacity (DC=1.0000). Remarkably, 13 rapidly mutating Y-STRs exhibited discriminatory power comparable to 44 moderate-to-slow loci, effectively distinguishing close relatives. Y-SNP analysis identified 137 terminal haplogroups with a haplogroup diversity of 0.9910, confirming high resolution for Chinese populations. By simultaneously analyzing recent familial relationships and deep phylogenetic lineages, this panel resolves Y-STR mutation ambiguities, offering a robust tool for paternal lineage and ancestry analysis in Chinese forensic applications.