Comparative Evaluation of Massively Parallel Sequencing STR kits with the Emphasis on Mixture Deconvolution Utilizing Probabilistic Genotyping.

The basis of individual identification in modern forensics is DNA typing of short tandem repeats (STRs).  This technique has brought a standardized, quantitative method with strong statistical underpinnings to the criminal justice system that has dramatically improved just and impartial outcomes.  While the fundamental principles behind STR typing have not changed, newly developed instrumentation and informative markers have the potential to address limitations of current techniques.

Current DNA analysis methods for individual identification have technical and throughput limitations. These methods are based on capillary electrophoretic sizing of selected amplicons, some of which would not be accepted today due to poor population frequency distributions or PCR amplification problems. Capillary electrophoresis (CE) is a single reaction detection method without capabilities for multiplexing and is consequently slow.  It only measures amplicon lengths and fails to detect sequence specific information that could improve STR analysis.  Finally, it is approaching the maximum number of STRs it can process, thus abandoning improvements from additional genetic markers.

The forensic community has begun to evaluate massively parallel sequencing (MPS) to overcome these problems.  Such instruments not only add additional sequencing information but have a nearly unlimited capacity for additional STRs and other genetic markers, thereby enhancing individual identification. As the sample separation is based on sequence information this allows for smaller amplicons, thus improving the outcome of degraded DNA. These instruments have the potential for significant improvements in throughput at lower costs.

The goal of this application is to evaluate two comparable MPS-STR kits, the ForenSeq™ MainstAY and the PowerSeq® 46GY System, by focusing on DNA mixture deconvolution. To achieve this goal, two Specific Aims are proposed: i) analysis of single-source samples, and ii) analysis of mixed samples. This will determine the limitations of the two MPS-STR kits regarding accuracy, sensitivity, levels of DNA degradation, and throughput. Data from this extensive testing will also be used to validate the research version of the fully continuous probabilistic genotyping software STRmix NGS. The new software is built on the foundation of the well-established and peer reviewed STRmix™. Expected products will aid in the forensic DNA interpretation from MPS generated data and will lead to more standardization by reducing subjectivity. Therefore, this project is substantial for forensic laboratories to implement MPS-STR technology in their routine casework.

This application directly addresses important research needs of the solicitation that can be found at the Organization of Scientific Area Committees (OSAC) website.