Development of PCR-Free DNA Typing Strategies via Real-Time High Throughput Sequencing

As submitted by the proposer: PCR amplification and capillary electrophoresis utilizing laser-excited fluorophores have been standard approaches for STR fragment analysis and mitochondrial DNA (mtDNA) sequencing in forensic testing for over 15 years. When challenged samples, relationship testing or lineage confirmation is needed, forensic casework often requires the use of testing beyond the suite of routinely typed STRs, including Y-STR and mtDNA analyses. A single forensic sample may require three individual processes, varying greatly in their methodologies, data analysis, and statistical approaches, to complete testing. Studies have shown that the evaluation of polymorphisms within the STR sequences and the full mitochondrial genome can improve discriminatory power of a forensic comparison; however, effective testing methodologies and analysis pipelines are, as of yet, not refined. The objective of this project is to develop and validate a PCR-free deep sequencing strategy targeting forensic microsatellite loci (autosomal-, Y- and X-STRs) and the complete mtGenome integrated with data analysis software. This strategy does not require amplification nor utilize current synthesis-based sequencing methods. This process will entail mining forensic marker regions from samples using optimized RNA baits, developed through collaboration with researchers at MYcroarray, similar to those successfully utilized in ancient DNA studies. The captured DNA sequences are then processed using novel direct-read sequencing methods developed by Oxford Nanopore Technologies in which the native DNA is directly interrogated with a portable sequencing system. With this approach, we can quantitatively assess the genetic composition of forensic samples with greater coverage than is currently available for forensic DNA testing. The optimized system will be evaluated for sensitivity, reproducibility, and concordance to fragment-based results, as well as test the ability to detect and deconvolute sample mixtures from deep coverage sequence data. A streamlined data analysis pipeline for processing the sequencing results will be established to provide not only the allele and haplotype results of the samples, but quality assessment information that can be used to determine the appropriate strategy for quality assurance measures and appropriate control structure for these types of analyses. This project contains a research and/or development component, as defined in applicable law. ca/ncf