In forensic analysis predictive tests for external visible characteristics (or 
EVCs), including inference of iris color, represent a potentially useful tool to 
guide criminal investigations. Two recent studies, both focused on forensic 
testing, have analyzed single nucleotide polymorphism (SNP) genotypes underlying 
common eye color variation (Mengel-From et al., Forensic Sci. Int. Genet. 4:323 
and Walsh et al., Forensic Sci. Int. Genet. 5:170). Each study arrived at 
different recommendations for eye color predictive tests aiming to type the most 
closely associated SNPs, although both confirmed rs12913832 in HERC2 as the key 
predictor, widely recognized as the most strongly associated marker with blue 
and brown iris colors. Differences between these two studies in identification 
of other eye color predictors may partly arise from varying approaches to 
assigning phenotypes, notably those not unequivocally blue or dark brown and 
therefore occupying an intermediate iris color continuum. We have developed two 
single base extension assays typing 37 SNPs in pigmentation-associated genes to 
study SNP-genotype based prediction of eye, skin, and hair color variation. 
These assays were used to test the performance of different sets of eye color 
predictors in 416 subjects from six populations of north and south Europe. The 
presence of a complex and continuous range of intermediate phenotypes distinct 
from blue and brown eye colors was confirmed by establishing eye color 
populations compared to genetic clusters defined using Structure software. Our 
study explored the effect of an expanded SNP combination beyond six markers has 
on the ability to predict eye color in a forensic test without extending the SNP 
assay excessively - thus maintaining a balance between the test's predictive 
value and an ability to reliably type challenging DNA with a multiplex of 
manageable size. Our evaluation used AUC analysis (area under the receiver 
operating characteristic curves) and naïve Bayesian likelihood-based 
classification approaches. To provide flexibility in SNP-based eye color 
predictive tests in forensic applications we modified an online Bayesian 
classifier, originally developed for genetic ancestry analysis, to provide a 
straightforward system to assign eye color likelihoods from a SNP profile 
combining additional informative markers from the predictors analyzed by our 
study plus those of Walsh and Mengel-From. Two advantages of the online 
classifier is the ability to submit incomplete SNP profiles, a common occurrence 
when typing challenging DNA, and the ability to handle physically linked SNPs 
showing independent effect, by allowing the user to input frequencies from SNP 
pairs or larger combinations. This system was used to include the submission of 
frequency data for the SNP pair rs12913832 and rs1129038: indicated by our study 
to be the two SNPs most closely associated to eye color.