Validation of Illumina's MiSeq FGx NGS Platform for Transition to Casework

The basis of individual identification in modern forensics is DNA typing by short tandem repeats (STR). 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, new instrumentation and informative biological markers developed over the past few years have the potential to address the limitations of current techniques as well as the need for increased throughput at lower costs. Current DNA analysis methods for individual identification have technical, cost 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 of PCR amplification problems. Capillary electrophoresis (CE) itself has inherent limitations. It is a single reaction detection method without capabilities for multiplexing and is consequently slow. It can only measure amplicon lengths and consequently fails to detect sequence specific information that could improve STR analysis. Finally it is approaching the maximum number of STRs it can process, thus effectively abandoning improvement from new STRs and SNPs that could improve individual identification, mixture deconvolution and hereditary analysis – at least in a single system. The forensic community is beginning to evaluate next generation sequencing (NGS) as a means to overcome these problems. Such instruments not only add additional sequencing information, but have a nearly unlimited capacity for additional STRs as well as single nucleotide polymorphisms (SNPs) markers, thereby enhancing individual identification. These instruments also have the potential for significant improvements in throughput at lower costs. The goal of this application is to validate the Illumina MiSeq FGx forensic sequencing platform with the aim of transitioning our laboratory from CE to NGS. To achieve these ends four Specific Aims are proposed: i) To compare NGS detection sensitivity, precision and accuracy against our current CE platform, ii) To evaluate NGS’s ability to improve mixture deconvolution through use of STRs and SNPs, iii) To compare outcomes of NGS and CE using degraded DNA, and iv) To evaluate throughput and costs between to the two systems. We believe this application directly addresses the goals of the solicitation as it will “result in the production of a validated method that may be replicated in other laboratories”, and will “have a direct and immediate impact on laboratory efficiency and assist in making laboratory policy decisions.” ca/ncf