Optimization of the Promega PowerSeq™ Auto/Y system for efficient integration within a forensic DNA laboratory.

The application of massively parallel sequencing (MPS) is growing in the forensic DNA field, as forensic DNA laboratories are continuously seeking methods to gain information from a limited or degraded forensic sample. However, the laborious nature of current MPS methodologies required for successful library preparation and sequencing leave opportunities for improvement to make MPS a practical option for processing forensic casework. In this study, the Promega PowerSeq™ Auto/Y System Prototype, a MPS laboratory workflow that incorporates multiplex amplification, was selected for optimization with the objectives to introduce automation for relieving manual processing, and to reduce the number of steps recommended by the standard protocol. Successful changes in the optimized workflow included a switch from column-based PCR purification to automatable bead-based purification, adoption of the library preparation procedures by a liquid handling robot platform, and removal of various time-consuming quality checks. All data in this study were found to be concordant with capillary electrophoresis (CE) data and previously-generated MPS results from this workflow. Read abundance and allele balance, metrics related to sample interpretation reliability, were not significantly different when compared to samples processed with the manufacturer's protocol. All the modifications implemented resulted in increased laboratory efficiency, reduced the protocol steps associated with risk of contamination and human error events, and decreased manual processing time by approximately 12h. These findings provide forensic DNA laboratories a more streamlined option when considering implementation of a MPS workflow.

HLA DQA1 and Polymarker validations for forensic casework: standard specimens, reproducibility, and mixed specimens.

This study describes the testing performed by the Minnesota Forensic Science Laboratory (MFSL) to validate the Amplitype DQA1 and Amplitype Polymarker (PM) PCR Amplification and Typing Kits before implementation for casework. All studies were based on the analysis of mock forensic case samples, which were assembled from various biological samples from individuals at the MFSL. To address the validation of standard specimens, DNA was isolated from semen, vaginal secretions, saliva, urine, and blood samples. Typing results from all tissues from a particular individual yielded the same typing results using both the DQA1 and PM systems. Reproducibility between laboratories was evaluated by having duplicate samples analyzed by a second laboratory. The Roche Biomedical Laboratories (RBL) were sent a duplicate set of mock cases and all analyses including extraction, quantitation, amplification, and typing were performed at the RBL using their established testing procedures. All typing results for both laboratories, from the approximate 30 single source samples analyzed, were in agreement. Mixed specimens were evaluated by examining the results obtained from semen/vaginal, semen/saliva, semen/blood, semen/ urine, and semen/vaginal/blood mixtures. All typing results of these mixtures for both laboratories were in agreement. It was determined that by incorporating a wash step of the sperm cell pellet, a complete separation of the nonsperm cell fraction was more likely to be attained. After completing the above studies, as well as population studies, environmental insult studies, and proficiency testing, the MFSL determined that both kits were suitable for use on forensic casework.

Validation studies for the genetic typing of the D1S80 locus for implementation into forensic casework.

A series of validation experiments were designed to evaluate, according to the Technical Working Group on DNA Analysis Methods (TWGDAM) guidelines, the analysis of the D1S80 locus for casework implementation. Approximately 400 samples from three different populations (Minnesota Caucasian, Minnesota African Americans, and Minnesota Native Americans) were typed to determine allele frequencies. Simulated forensic type specimens (blood, saliva, hair and semen, or vaginal secretions) were typed to demonstrate that deoxyribonucleic acid (DNA) extracted from various tissues of an individual yield the same D1S80 type. Dilution studies were performed and it was determined that a wide range of input DNA (0.5 ng to 40.0 ng) will consistently yield typeable results. The evaluation of DNA from various animals showed that the D1S80 locus is specific to human DNA within the limits of the parameters tested. The reproducibility of the system was tested by duplicate analysis of approximately 200 population samples. Duplicate samples were analyzed on both horizontal and vertical gel systems. In addition, simulated forensic specimens were analyzed by two independent laboratories: the Minnesota Forensic Science Laboratory (MFSL) and the Roche Biomedical Laboratories (RBL). All analyses, including extraction, quantitation, amplification and typing, were performed independently. All typing results for both laboratories were in agreement. By the analysis of mixtures from various simulated casework type mixtures, it was demonstrated that the D1S80 typing system is suitable for analyzing mixtures. In addition to the simulated casework, evidentiary samples from several adjudicated cases previously analyzed by restriction fragment length polymorphism (RFLP) analysis and/or DQA1 were typed at the D1S80 locus. The D1S80 results were consistent with previous RFLP and/or DQA1 results regarding inclusions/exclusions.