Forensic DNA Phenotyping (FDP), i.e. the prediction of human externally visible traits from DNA, has become a fast-growing subfield within forensic genetics due to the intelligence information it can provide from DNA traces. FDP outcomes can help focus police investigations in search of unknown perpetrators, who are generally unidentifiable with standard DNA profiling. The FDP system tested in the current study consists of two SNaPshot-based multiplex assays targeting a total of 41 SNPs via a novel multiplex assay for 17 skin color predictive SNPs and the previous HIrisPlex assay for 24 eye and hair color predictive SNPs, 19 of which also contribute to skin color prediction. The HIrisPlex-S system further comprises three statistical prediction models, the previously developed IrisPlex model for eye color prediction based on 6 SNPs, the previous HIrisPlex model for hair color prediction based on 22 SNPs, and the recently introduced HIrisPlex-S model for skin color prediction based on 36 SNPs. In the forensic developmental validation testing, the novel 17-plex assay performed in full agreement with the Scientific Working Group on DNA Analysis Methods (SWGDAM) guidelines, as previously shown for the 24-plex assay. Sensitivity testing of the 17-plex assay revealed complete SNP profiles from as little as 63 pg of input DNA, equaling the previously demonstrated sensitivity threshold of the 24-plex HIrisPlex assay. Testing of simulated forensic casework samples - such as blood, semen, saliva stains - of inhibited DNA samples of low quantity touch (trace) DNA samples, and artificially degraded DNA samples, as well as concordance testing, demonstrated the robustness, efficiency, and forensic suitability of the new 17-plex assay, as previously shown for the 24-plex assay. Finally, this article provides an update to the publicly available HIrisPlex website https://hirisplex.erasmusmc.nl/, now allowing the estimation of individual probabilities for three eye, four hair, and five skin color categories from HIrisPlex-S input genotypes. The HIrisPlex-S DNA test represents the first forensically validated tool for skin color prediction, and reflects the first forensically validated tool for simultaneous eye, hair, and skin color prediction from DNA. (publisher abstract modified)
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