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/microL of DNA within a factor of 0.3. Extracts were amplified in triplicate with AmpFlSTR 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.