An evaluation of ForenSeq DNA Signature Prep iiSNP mixture interpretation using a probabilistic genotyping method

The analysis of autosomal short tandem repeat markers (STRs) using capillary electrophoresis (CE) technology remains the dominant method for forensic investigations globally. However, next-generation sequencing technologies are increasingly being adopted as they enable simultaneous amplification of both STR and SNP (single-nucleotide polymorphisms) loci in large panels, amongst other benefits. Unlike STRs, which are highly variable or polymorphic, the majority of SNPs are biallelic, meaning there are only two allelic variants. A single SNP marker on its own is unlikely to provide as much evidentiary value as a single STR marker. However, the interpretation of SNP markers from mixed DNA evidence is computationally simpler than that of STR markers because they do not produce stutter artefacts and, being biallelic, there are fewer possible genotype combinations. SNPs are increasingly recognised as valuable markers, complementary to STRs, and have been proven to be useful for applications including kinship analyses, informing ancestry, providing phenotypic characters for investigative leads, or forensic investigative genetic genealogy. The most common approach for the interpretation of autosomal STR (aSTR) profiles developed using CE technology within the US, UK, and Australasia is now based on probabilistic genotyping methods. Some of these published and tested models have been applied to aSTR profiles developed using NGS technologies and in this paper, we investigate the application of one such model (STRmix™ NGS) to the interpretation of mixed SNP profiles. Comparisons of likelihood ratios (LRs) assigned to donors and non-donors show expected trends in single-source profiles. However, in more complex mixtures, the system's ability to differentiate between true and false donors diminishes quickly. This is consistent with theoretical limitations for biallelic markers. While this study is limited to the 94 iiSNPs (identity informative SNPs) included in the ForenSeq kit, the findings suggest that simple SNP profiles can be interpreted within the existing STRmix™ NGS framework. This provides another potential tool for SNP profile analysis and lays the foundation for future joint interpretation of STR and SNP markers, or assays with more SNP markers.