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Prediction of eye and hair colour from DNA can be an important investigative tool in forensic cases if conventional DNA profiling fails to match DNA from any known suspects or cannot obtain a hit in a DNA database. The HIrisPlex model for simultaneous eye and hair colour predictions was developed for forensic usage. To genotype a DNA sample, massively parallel sequencing (MPS) has brought new possibilities to the analysis of forensic DNA samples. As part of an in-house validation, this study presents the genotyping and predictive performance of the HIrisPlex SNPs in a Norwegian study population, using Verogen's ForenSeq™ DNA Signature Prep Kit on the MiSeq FGx system and the HIrisPlex webtool. DNA-profiles were successfully typed with DNA input down to 125 pg. In samples with DNA input < 125 pg, false homozygotes were observed with as many as 92 reads. Prediction accuracies in terms of AUC were high for red (0.97) and black (0.93) hair colours, as well as blue (0.85) and brown (0.94) eye colours. The AUCs for blond (0.72) and brown (0.70) hair colour were considerably lower. None of the individuals was predicted to have intermediate eye colour. Therefore, the error rates of the overall eye colour predictions were 37% with no predictive probability threshold (pmax) and 26% with a probability threshold of 0.7. We also observed that more than half of the incorrect predictions were for individuals carrying the rs12913832 GG genotype. For hair colour, 65% of the individuals were correctly predicted when using the highest probability category approach. The main error was observed for individuals with brown hair colour that were predicted to have blond hair. Utilising the prediction guide approach increased the correct predictions to 75%. Assessment of phenotype-genotype associations of eye colours using a quantitative eye colour score (PIE-score), revealed that rs12913832 AA individuals of Norwegian descent had statistically significantly higher PIE-score (less brown eye colour) than individuals of non-northern European descent. To our knowledge, this has not been reported in other studies. Our study suggests that careful assessment of the target population prior to the implementation of forensic DNA phenotyping to case work is beneficial. • All 24 HIrisPlex SNPs performed well with DNA input as low as 125 pg. • Eye colour of 59–63% of the individuals was predicted correctly (model-depending). • Most individuals with incorrectly predicted eye colour carried rs12913832 GG. • Norwegians with rs12913832 AA had less pigmented eyes than non-northern Europeans. • Hair colour of 65–75% of the individuals was predicted correctly (model-depending). [ABSTRACT FROM AUTHOR]

Many genetic variants with association to normal pigmentary variation have been identified. Several assays for prediction of pigmentary traits have been developed using various types of genotyping methods, including Single Base Extension (detected by CE or MALDI-TOF MS) and real-time PCR. To circumvent the limited multiplexing capacity of these methods, we designed the Human Pigmentation (HuPi) AmpliSeq™ custom panel for the Ion S5™ System. The panel was designed as one large multiplex PCR using the Ion AmpliSeq™ Designer v7.0.6 (Thermo Fisher Scientific). The PCR amplifies 183 genetic variants with association to normal pigmentary variation of the eyes, skin, and hair in 46 genes including OCA2 , MC1R , SLC24A4, SLC45A2, TYR , ASIP , HERC2 , and TYRP1. The HuPi panel consists of 161 amplicons with an average amplicon size of 118 bp (70–137 bp), which makes the panel applicable for forensic casework. For the initial evaluation of the panel, we successfully typed 68 individuals. Fifteen loci were excluded due to low or no coverage (<30x). The heterozygote balances were around 1 for most loci, and the median level of noise <1%. Profiles with 168 loci were obtained from only 100 pg input DNA. In conclusion, the HuPi panel is a promising new assay for typing of pigmentary markers. [ABSTRACT FROM AUTHOR]

• Pigmentation phenotypes and underlying genotypes are highly correlated. • Previous genetic prediction models did not consider phenotype correlations. • Testing impact of pigmentation trait correlation on genetic pigmentation prediction. • Observed correlated pigmentation phenotypes improve genetic pigmentation prediction. • DNA-predicted correlated phenotypes have no impact on genetic pigmentation prediction. Predicting appearance phenotypes from genotypes is relevant for various areas of human genetic research and applications such as genetic epidemiology, human history, anthropology, and particularly in forensics. Many appearance phenotypes, and thus their underlying genotypes, are highly correlated, with pigmentation traits serving as primary examples. However, all available genetic prediction models, including those for pigmentation traits currently used in forensic DNA phenotyping, ignore phenotype correlations. Here, we investigated the impact of appearance phenotype correlations on genetic appearance prediction in the exemplary case of three pigmentation traits. We used data for categorical eye, hair and skin colour as well as 41 DNA markers utilized in the recently established HIrisPlex-S system from 762 individuals with complete phenotype and genotype information. Based on these data, we performed genetic prediction modelling of eye, hair and skin colour via three different strategies, namely the established approach of predicting phenotypes solely based on genotypes while not considering phenotype correlations, and two novel approaches that considered phenotype correlations, either incorporating truly observed correlated phenotypes or DNA-predicted correlated phenotypes in addition to the DNA predictors. We found that using truly observed correlated pigmentation phenotypes as additional predictors increased the DNA-based prediction accuracies for almost all eye, hair and skin colour categories, with the largest increase for intermediate eye colour, brown hair colour, dark to black skin colour, and particularly for dark skin colour. Outcomes of dedicated computer simulations suggest that this prediction accuracy increase is due to the additional genetic information that is implicitly provided by the truly observed correlated pigmentation phenotypes used, yet not covered by the DNA predictors applied. In contrast, considering DNA-predicted correlated pigmentation phenotypes as additional predictors did not improve the performance of the genetic prediction of eye, hair and skin colour, which was in line with the results from our computer simulations. Hence, in practical applications of DNA-based appearance prediction where no phenotype knowledge is available, such as in forensic DNA phenotyping, it is not advised to use DNA-predicted correlated phenotypes as predictors in addition to the DNA predictors. In the very least, this is not recommended for the pigmentation traits and the established pigmentation DNA predictors tested here. [ABSTRACT FROM AUTHOR]

Abstract: Predictions of human traits from biological stains with genetic methods have recently gained tremendous interest in criminal investigations. Various studies have revealed that single nucleotide polymorphisms (SNPs) within the HERC2, OCA2, MC1R, SLC24A5, SLC45A2 and TYR genes have been strongly associated with pigmentation trait variations in Caucasian populations. The prediction probability estimation for eye and hair color in the investigation conducted on the Slovenian population is associated mostly with two HERC2 SNPs: rs1129038 and rs12913832, with a probability value of 0.96, as has also been indicated in previous investigations. Other SNPs have a small additional affect on the prediction of eye color (OCA2 – rs1667394, rs7170989, TYR – rs1393350, and SLC45A2 – rs16891982) or hair color (OCA2 – rs1667394, rs7495174, rs7170989, MC1R – rs1805005, and TYR – rs139335). The variation in the rest of the SNPs (MC1R – rs1805008, OCA2 – rs1800407, SLC45A2 – rs26722, and SLC24A5 – rs1426654), are not as meaningful in terms of the prediction of values for eye and hair color. The SIplex assay is designed to be very sensitive, in order to indicate all twelve SNPs genotypes with only 62.5pg of DNA. Using twelve SNPs, eight of which being more or less usable for eye and hair color prediction based on the human genotype, we managed to develop a sensitive and reliable multiplex genotyping assay. [Copyright &y& Elsevier]