The MASTiFF panel-a versatile multiple-allele SNP test for forensics

Forensic identification tests often need recourse to markers that can successfully type highly degraded DNA, and binary single nucleotide polymorphisms (SNPs) have become the variants of choice for such analyses because of their short amplified fragment lengths. The two main drawbacks of SNPs are their reduced power of discrimination per marker compared with mainstream forensic STRs and an inability to robustly detect mixed DNA-particularly using capillary electrophoresis genotyping systems such as SNaPshot™, where the dye signals are much more imbalanced than those of STR profiles. This study compiled a compact set of multiple-allele SNPs consisting of loci that had three or four nucleotide variants at the same site in order to address the lack of mixture detection capability with binary SNP tests, as well as improving levels of polymorphism per SNP by transitioning to a maximum of six or ten genotypes per locus. We report the development and optimisation of a SNaPshot-based forensic test comprising 27 tri-allelic and 2 tetra-allelic SNPs, which we named MASTiFF: a multiple-allele SNP test for forensics. Assessments of the MASTiFF panel's levels of discrimination power in the five main population groups indicate random match probabilities ranging from 10^-15 down to 10^-20-improving the levels possible from an equivalent number of binary SNPs. The SNaPshot test was able to detect simple mixtures successfully with more than two alleles observed in 30% of SNPs. From allele frequency data, it is estimated that more than two alleles will be present in at least one MASTiFF SNP in 99.8% of two-person mixtures, making this panel an ideal supplementary test when SNPs are chosen for the analysis of degraded forensic DNA.