Revealing the challenges of low template DNA analysis with the prototype Ion AmpliSeq™ Identity panel v2.3 on the PGM™ Sequencer

• STR fragments obtained with the NGM SElect™ Kit and SNPs using a prototype version of the Ion AmpliSeq™ Identity panel (v2.3) with massive parallel sequencing was evaluated on LtDNA samples.
• Sensitivity study was performed down to 5 pg with control-DNA and degradation studies were conducted with control DNA and blood reference samples. A total of 1900 independent genotypes were analyzed. Allele balance and consistency in the obtained genotypes were evaluated.
• Casework samples were processed with Ion AmpliSeq™ Identity panel and Random Match Probability (RMP) was calculated in order to compare the Power of Identity (PI) for both the SNP and STR profiles.
• On the basis of our evaluation, the prototype Ion AmpliSeq™ Identity panel v2.3 demonstrated to be a fast, robust and valuable tool for gathering identity information in challenging situations such as LtDNA.

Forensic scientists frequently have to deal with the analysis of challenging sources of DNA such as degraded and low template DNA (LtDNA). The capacity to genotype difficult biological traces has been facilitated by emerging technologies. Massive parallel sequencing (MPS) on microchip among other technologies promises high sensitivity and discrimination power. In this study we evaluated the combined use of the Quantifiler® Trio DNA Quantification Kit with the prototype Ion AmpliSeq™ Identity panel v2.3 and PGM™ platform in LtDNA samples. Coverage, allele balance, allele drop-out/in, consistency and variance were assessed. Overall, the results showed a great level of performance and consistency in terms of genotyping capability even under the most challenging conditions, making it possible to obtain consistent SNP profiles with 31 pg of DNA and partial informative profiles with as little as 5 pg or with severely degraded DNA. In addition, we demonstrated that the stochastic effects observed in some samples are due to the amplification of the library rather than sequencing.Based on our data, we proposed general recommendations for the analysis of casework samples starting from the use of quantification data, which proved to be critical in deciding whether to process the samples via STR (short tandem repeat) analysis or SNP MPS.In our experience, the use of the prototype Ion AmpliSeq™ Identity panel v2.3 has revealed a new applicable solution for processing LtDNAs. This approach provides users with an additional tool for analysis of traces that either would not give informative results with conventional STR-based techniques.