Estimating the number of contributors to two-, three-, and four-person mixtures containing DNA in high template and low template amounts

Aim To develop guidelines to estimate the number of contributors to two-, three-, and four-person mixtures containing either high template DNA (HT-DNA) or low template DNA (LT-DNA) amounts. Methods Seven hundred and twenty-eight purposeful two-, three-, and four-person mixtures composed of 85 individuals of various ethnicities with template amounts ranging from 10 to 500 pg were examined. The number of alleles labeled at each locus and the number of labeled different and repeating alleles at each locus as well over all loci for 2 HT-DNA or 3 LT-DNA replicates were determined. Guidelines based on these data were then evaluated with 117 mixtures generated from items handled by known individuals. Results The number of different alleles over all loci and replicates was used to initially categorize mixtures. Ranges were established based on the averages plus and minus 2 standard deviations, and to encompass all observations, the maximum and the minimum values. To differentiate samples that could be classified in more than one grouping, the number of loci with 4 or more repeating or different alleles, which were specific to three- and four-person mixtures, were verified. Misclassified samples showed an extraordinary amount of allele sharing or stutter. Conclusions These guidelines proved to be useful tools to distinguish low template and high template two-, three-, and four-person mixtures. Due to the inherent higher probability of allele sharing, four-person mixtures were more challenging. Because of allelic drop-out, this was also the case for samples with very low amounts of template DNA or extreme mixture ratios.