Systematic evaluation of two-person DNA mixtures using the precision ID GlobalFiler™ NGS STR panel v2: A focus on...

The analysis of DNA mixtures remains a challenging task in forensic genetics, as the presence of multiple contributors and highly imbalanced DNA ratios complicate genotyping and interpretation. Next-generation sequencing (NGS) improves resolution by detecting both length- and sequence-based polymorphisms, potentially enhancing mixture deconvolution. Here, we systematically evaluated the performance of the Precision ID GlobalFiler™ NGS STR Panel v2 for two-person DNA mixtures. Three DNA standard materials (9947 A, 9948, and 2800 M) were used to construct pairwise two-person mixtures at eight different mixing ratios (from 1:1 to 1:99), with triplicate sequencing. Key sequencing performance metrics (total reads; depth of coverage, DoC; on-target rates; locus balance; allele balance, A_b) and detection rate of minor contributor-specific alleles (DR_MSA) were comprehensively assessed. Results showed robust performance, with mean DoC ranging from 4419× to 6616× and on-target rates consistently above 89.50%. The DR_MSA gradually decreased as the proportion of the minor contributor diminished, with trace minor contributor-specific alleles still detectable at the extreme ratio of 1:99, outperforming the recently reported nanopore sequencing-based MiniHap panel; slight inter-combination differences in DR_MSA suggested genetic background impacts on mixture deconvolution efficiency. Furthermore, we characterized the relationship between mixing ratios and A_b variation across different genotype patterns, revealing that A_b variation trends were strongly influenced by contributor zygosity and the number of shared alleles. This systematic evaluation provided empirical evidence and practical guidelines for applying this NGS Panel to forensic two-person DNA mixture deconvolution, and lay a foundation for optimizing probabilistic genotyping strategies for NGS-based STR mixture analysis.