Validation of YFiler Plus Amplification Kit for the San Diego Police Department

Currently, the San Diego Police Department Forensics Biology Unit utilizes the AmpFlSTR® YFiler PCR Amplification Kit from Applied Biosystems. However, they have been looking to upgrade to the new YFiler Plus Amplification Kit from Thermo Fisher Scientific because the current kit is outdated and YFiler Plus offers more discrimination power due to the increased number of markers and improved performance with degraded samples. Hence the two specific aims of this project were: 1) to perform a validation by conducting multiple studies to compare the efficacy of the new typing kit versus the current typing kit, 2) document and write reports of validation results from each study performed. The validation began with finding the optimal amplification conditions on the Veriti (Thermalcycler) and finding the best parameters for capillary electrophoresis on the 3500 Genetic Analyzer. Afterwards, the following studies were carried out: baseline, sensitivity, stutter, balance, concordance, stochastic, and DNA mixtures. Once the validation was finished, a summary was written for each study to elaborate on how and why and the experiments were carried out, discuss results and analysis, and present meaningful conclusions. Once the expectations were met, the new kit was implemented into forensic casework. The forensic biology unit currently employs the AmpFlSTR® YFiler PCR Amplification Kit, a kit that was implemented and validated in May 2007. The forensic biology unit decided to validate the YFiler Plus Amplification Kit as a potential replacement as it promises to provide an improved baseline and higher recovery of alleles. The YFiler PCR Amplification Kit amplifies the same seventeen loci markers that are in the Yfiler Plus Kit (DYS456, DYS389I, DYS390, DYS389II, DYS458, DYS19, DYS385 a/b, DYS393, DYS391, DYS439, DYS635, DYS392, YGATAH4, DYS437, DYS438, and DYS448). Upon verifying the amplification of these markers, the ten additional Y-STR loci (DYS576, DYS627, DYS460, DYS518, DYS570, DYS449, DYS481, DYS387S1 a/b, and DYS533) included in the Yfiler Plus Kit will be tested. These new markers are important as they offer greater discrimination when searching for the male suspect, which increases the statistical probability of obtaining the correct identification. Overall, the YFiler Plus Amplification Kit from Thermo Fisher Scientific is a 6-dye multiplex assay that allows for amplification of 27 Y-STR markers. It is created for forensic laboratories that carry out challenging sexual assault cases and it optimizes the analytical processes within forensic laboratory casework. With YFiler Plus Kit, the following features resulted: markers that mutate more frequently and amplify more efficiently, improved DNA results from degraded samples that contain PCR inhibitors, increased genotyping accuracy through the developed allelic ladder and virtual bins, and enhanced protocol efficiency. Even in the presence of an abundance of female DNA, the new Yfiler Plus Kit readily detected male DNA from mixed samples.