Droplet digital PCR: assessing the increased performance for DNA quantification in forensic science

DNA quantification is a vital and required step for processing forensic biology evidence. While nearly all forensic laboratories quantify nuclear and mitochondrial DNA using quantitative PCR (qPCR) assays, the qPCR method in general has two shortcomings: 1) a standard curve is used to determine DNA copy number in a sample, which provides indirect quantification and is sensitive to reproducibility issues, and 2) amplification can be negatively impacted by sample-associated inhibitors. Newer quantification methods, such as droplet digital PCR (ddPCR) can address these shortcomings, while remaining directly compatible with existing qPCR assays. In ddPCR, a single 20 µL reaction volume is partitioned into approximately 20,000 individual 1 nL reactions prior to PCR. Post PCR analysis to detect the presence of target amplicon in each droplet allows for absolute quantification of the target DNA template in the initial reaction mix. Additionally, by partitioning all reaction components, including PCR inhibitors, ddPCR minimizes the impact of such inhibitors, a feature of the process that allows analysis of specimens burdened with high inhibitor concentrations to be analyzed where traditional qPCR would fail. This proposed study is focused on assessing the increased performance that ddPCR provides over qPCR for DNA quantification in forensic science. To achieve this, a single 5-plex quantification assay will be developed utilizing previously published qPCR targets: nuclear DNA (short and long autosomal, Y-chromosome), mitochondrial DNA (short) and an internal positive control. Side-by-side experiments will be completed by two separate analysts on qPCR and ddPCR instruments to assess a) sensitivity and accuracy by quantifying serial dilutions of high-quality DNA samples, b) performance in the presence of previously reported inhibitors of DNA amplification, and c) performance with mock evidence samples that likely possess degraded DNA. To evaluate the predictability of ddPCR quantification and calculated degradation index to the success of traditional DNA analyses, STR typing and mitochondrial whole control region sequencing will be completed on all inhibition and mock evidence samples. Statistical analyses will be completed to establish whether a significant difference in the number of copies for the five targets is noted between qPCR and ddPCR. We hypothesize that ddPCR will outperform qPCR, providing a strong framework to justify forensic laboratories transitioning to completing DNA quantification using ddPCR.        At least two peer-reviewed scientific publications will result from this study, and findings will be disseminated with the community via presentations at scientific conferences and open forum webinars. CA/NCF