Development and Preliminary Evaluation of a Nanopore-Based 133-Locus MiniHap Panel for Forensic DNA Mixture Interpretation

Background: Forensic DNA mixture interpretation relies on the detection of contributor-specific alleles and sufficiently informative markers to distinguish between competing propositions. MiniHap combines multiple single nucleotide polymorphisms (SNPs) distributed within relatively compact genomic regions into highly polymorphic multiallelic haplotypes. Resolving these extended haplotypes requires the physical phase of their constituent SNPs to be preserved, for which nanopore long-read sequencing offers a distinct advantage by spanning an entire MiniHap within a single read, thereby potentially providing additional information for the analysis of complex DNA mixtures. Methods: In this study, we developed a nanopore sequencing-based MiniHap panel. And nine artificial mixtures containing 2–4 contributors were evaluated for allele detection and likelihood ratio (LR) after filtering with a relative threshold of 0.05 of the total sequencing depth at each locus. Results: The resulting panel included 133 loci, with a mean effective number of alleles (Ae) of 4.37, 108 loci exhibiting Ae > 3, 39 loci exhibiting Ae > 5, and 2 loci exhibiting Ae > 10. In balanced multi-person mixtures, the panel performed well and maintained clear discrimination between the designated contributor and non-contributors, even in the four-person balanced mixture (log10(LR) = 34.97). In contrast, unbalanced mixtures showed substantial loss of alleles unique to the minor contributor, with only 8–21% detected and corresponding log10(LR) values below 0. This may reflect the reduced effective information content resulting from minor-component dropout and sequencing noise. Conclusions: In conclusion, this panel supports the analysis of balanced multi-person mixtures but has limited applicability to highly unbalanced mixtures under the fixed filtering threshold used in this study. Further work may focus on optimizing filtering strategies and characterizing platform-specific factors affecting DNA mixture analysis.