DNA Quantitation and Next Generation Sequencing Instrumentation for High Throughput Sexual Assault Evidence Processing, Generating Investigative Leads, and Enhanced Human Identification.
Abstract
The grant proposal submitted to the Defense University Research Instrumentation Program (DURIP) by the PI requested funds to purchase a Life Technologies (ThernoFisher) 7500 real time PCR system (7500) and the Massively Parallel Sequencing (MPS) next generation sequencing platform Ion Torrent Personal Genome Machine (PGM) System. Only approximately one third of the funds were awarded. Internal funding efforts enabled the PI to purchase all the proposed instrumentation. Shortly after the grant was awarded a newer version of the PGM was commercialized by Thermofisher, the Ion SS, which was the one purchased with the DURIP funds. Current forensic DNA testing for human identification (HID) purposes is based on short tandem repeat (SIR) markers analyzed on capillary electrophoresis (CE) plat forms. The present technology, while robust and reliable, is limited in the number of markers that can be typed in a single reaction. Multiple assays can be employed to type different markers from the same sample, but this requires considerable time, resources, and amount of DNA. These factors presently limit the amount of information obtainable from forensic samples. The Ion S5 will be used to develop assays to simultaneously sequence thousands of forensically informative regions, providing, together with the conventional DNA profile, information on the biogeographic ancestry and physical traits of an individual, thus enhancing human identification efforts. Another research avenue that will be explored is that of DNA mixture analysis and interpretation. By enabling direct sequencing of STR markers, MPS arnows complex DNA mixture deconvolution more so than the current teclrnology. In fact, complex mixtures are the Achilles tendon of current forensic DNA methods. Factors that complicate their interpretation include but are not limited to, sample degradation, allele sharing between contributors, allele drop-out, and PCR artifacts such as stutter, particularly when the minor contributor is in the stutter peak height range of the major. Implementing mixture analysis software can increase consistency in the interpretation but cannot eliminate these factors. Allele drop, for example is a consequence of low/degraded template and stutter is an artifact of polymerase chain reaction (PCR) of STRs. MPS of STR alleles has been proposed as an option to address some of these issues. Yet experiments have shown that interpretation of STR sequences from NGS data is not as straight forward as originally anticipated, especially when dealing with imbalance mixtures. Microhaplotypes (MHs) are loci of two or more SNPs within a short distance from each other (<200 nucleotides i.e. micro ) with three or more allelic combinations ( haplotypes ). Conventional Sanger sequencing does not allow detennining the cis/trans relationship between individual SNP alleles (i.e. the haplotype). NGS methods instead, when SNPs are in the same amplicon, allow sequencing of individual strands and haploty-pe detection at a locus. One focus of the research conducted with the S5 is to (l) develop an MPS multiplex assay targeting MHs and selected HLA regions for mixture deconvolution; (2) to determine/confirm allele frequencies in the major US populations to enable sound implementation of the assay; (3) to evaluate its effectiveness on forensic samples. Using MPS technology to target small informative markers combined with the absence of stutter will enhance complex mixture deconvolution capabilities, much needed in the forensic DNA community. Accurately determining the quantity and quality of DNA in a forensic sample is paramount for a successful MPS analysis. This is accomplished with the 7500, which can determine the quantity of DNA, determine degradation levels, and detect inhibitors of downstream processes.
Document Details
- Document Type
- DoD Grant Award
- Publication Date
- Jan 12, 2017
- Source ID
- W911NF1510302
Entities
People
- Daniele Podini
Organizations
- Army Contracting Command
- George Washington University
- United States Army