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

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

Validation of Testing and Interpretation Protocols for Low Template DNA Samples Using AmpFℓSTR® Identifiler®

Aim To test the reliability, robustness, and reproducibility of short tandem repeat (STR) profiling of low template DNA (LT-DNA) when employing a defined set of testing and interpretation parameters. Methods DNA from known donors was measured with a quantitative real time polymerase chain reaction (PCR) assay that consistently detects less than 1 pg/μL of DNA within a factor of 0.3. Extracts were amplified in triplicate with AmpFSTR® Identifiler® reagents under enhanced PCR conditions. Replicates were examined independently and alleles confirmed using a consensus approach. Considering observed stochastic effects inherent to LT-DNA samples, interpretation protocols were developed and their accuracy verified through examination of over 800 samples. Results Amplification of 100 pg or less of DNA generated reproducible results with anticipated stochastic effects. Down to 25 pg of DNA, 92% or more of the expected alleles were consistently detected while lower amounts yielded concordant partial profiles. Although spurious alleles were sometimes observed within sample replicates, they did not repeat. To account for allelic dropout, interpretation guidelines were made especially stringent for determining homozygous alleles. Due to increased heterozygote imbalance, stutter filters were set conservatively and minor components of mixtures could not be resolved. Applying the resultant interpretation protocols, 100% accurate allelic assignments for over 107 non-probative casework samples, and subsequently 319 forensic casework samples, were generated. Conclusion Using the protocols and interpretation guidelines described here, LT-DNA testing is reliable and robust. Implementation of this method, or one that is suitably verified, in conjunction with an appropriate quality control program ensures that LT-DNA testing is suitable for forensic purposes