Evaluation and comparative analysis of direct amplification of STRs using PowerPlex® 18D and Identifiler® Direct...

Short tandem repeat (STR) analysis remains the primary forensic tool for DNA identification. Because of the success of forensic DNA typing and the use of database searches to develop investigative leads, there is an increased demand for populating forensic DNA databases. Reference samples tend to be of high quantity and quality and are somewhat standardized in format. Being more predictable in quality than unknown forensic casework samples, reference samples lend themselves to alternate methods of analysis such as direct amplification. Two commercially available direct amplification kits for processing reference samples were evaluated. The kits are PowerPlex(®) 18D (Promega Corp., Madison, Wisconsin) and Identifiler(®) Direct (Life Technologies, Carlsbad, CA). Both kits offer the core CODIS loci plus amelogenin, and the loci D2S1338, D19S433. The PP18D kit offers two additional loci, Penta E and Penta D. To determine the robustness and reliability of the PP18D and ID Direct amplification systems, buccal cell samples (deposited on FTA paper using the EasiCollect™ device (Florham Park, NJ)) from 400 anonymous donors were analyzed under conditions to achieve a high rate of successful typing. First-pass success rates were 96.25%, 96.25%, and 95% for PP18D with a 5s injection, ID Direct with a 10s injection, and ID Direct with a 5s injection, respectively. Profiles that could not be typed were not a result of the kits' performance but were a result of the inherent variation in the amount of DNA obtained with the collection device and buccal cells. Low signal profiles can be re-analyzed by either re-injecting for a longer time or by re-amplification with an additional PCR cycle. Overloaded profiles can be re-analyzed by re-injecting for a shorter time or by re-amplification with one less cycle. All called typing results, when interpretable, were consistent under the prescribed conditions, different injection times, and 26-28 PCR cycles for both chemistries. Peak height ratios for both kits were well balanced with peaks ranging in height >2000 RFUs to those with one or more peaks with heights <100 RFUs. A change in the ILS morphology sloping downward to the right relative to a normal ILS profile for PP18D and ID Direct was an indication of a poor injection. Re-injection effectively overcame the effect manifested by a sloping ILS phenomenon. A subset of samples were subjected to direct amplification using the reagents in Identifiler(®) Plus kit and successful typing results were obtained for the majority of samples. However, the profiles displayed increased amounts of non-adenylated products. The results of this study demonstrate that PP18D and ID Direct are both robust kits for direct amplification. The interpretation guidelines used for this study can form a basis for internal validation studies by databasing laboratories.