Technical reliability of genotyping SNPs for forensic DNA phenotyping using SNaPshot- and MPS-based assays

In recent years, several countries have undergone changes in their legal framework, now explicitly allowing the analysis of genetic markers for the purpose of forensic DNA phenotyping (FDP). Consequently, laboratory workflows for the analysis of appearance informative single nucleotide polymorphisms (SNPs) have been established in several laboratories. Currently, the HIrisPlex-S marker panel and webtool are the most widely used set of appearance informative SNP markers and statistical model used for phenotype predictions. However, many different laboratory protocols are employed for SNP genotyping, mostly using either massive parallel sequencing or single base extension technology. For the GEDNAP and TrACE proficiency tests, FDP modules were introduced in 2021 and 2023. These represented the first instances in which identical samples were analyzed by a fairly large number of laboratories, each of them employing their own laboratory-validated protocol. While mostly consistent phenotyping results were obtained, discrepant genotyping results were observed for some of the analyzed HIrisPlex-S-SNPs in BNC2, OCA2, and TYR. By performing a systematic in-silico analysis of commonly used primer sequences and sequencing the flanking regions of target SNPs in the affected samples from the collaborative exercises, we were able to identify primer binding site mutations, amplification of off-target products and overlap of SBE primers as risk factors for (analysis method-dependent) genotyping discrepancies. While the impact on phenotyping results was minor to negligible in all cases reported here, the issues uncovered by this in-depth analysis may provide a basis for improvements towards more consistent results in the future.