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<ul> <LI>Develop a "reverse search" tool, which would allow a sequence database generated by a massively parallel sequencing (MPS) run to be searched with a limited number of key pathogen sequences. This will cut processing time exponentially, increasing efficiency and throughput. <LI>Test MPS diagnostic capacity on select plant pathogens from viruses, phytoplasmas, bacteria, fungi and stramenopiles. <LI>Test MPS forensic (strain typing) capacity on select plant pathogens, where previous typing data is available.<LI> Test the ability of MPS to detect plant pathogens that have been genetically engineered to express toxins or proteins harmful to humans, by screening plant pathogen DNA samples spiked with examples of such genes or commonly used genetic engineering promoters and/or plasmid screening markers.<LI> Establish a collaborative research and educaitonal link between the Oklahoma State National Institute for Microbial Forensics & Food and Agricultural Biosecurity and the Fort Detrick National Interagency Biodefense Campus.
Non-Technical Summary: Diagnostics laboratories face tremendous challenges in dealing with plant pathogens. The vast array of possible crop species, each with their own set of possible pathogens, results in the need to be able to detect thousands of microbes with high specificity and sensitivity. In addition, the legal requirements for diagnostic labs require that all assays be validated and all assay users be certified in order for the results to be allowed as valid. Under these conditions, diagnostics labs would spend all of their time and resources in developing assays, validating those assays and training certifying workers. The best solution would be a single protocol that could detect and identify any and all pathogens in a given sample simultaneously, with the capacity to do forensic analysis and search for unknown and genetically modified organisms. No such technology currently exists, but MPS has the potential to fulfil such a role. The goal and objectives of this project are to develop MPS as a diagnostic tool that can meet all the needs described above. <P> Approach: Massively parallel sequencing (MPS) generates tremendous numbers of overlapping short sequence reads that are then compiled by computer to generate huge contigs of sequence. In theory, MPS generates sequence for all DNAs in a given sample. For example, if a plant is infected with a bacterial pathogen the resulting MPS data would contain a mixture of plant and bacterial sequences. The most common use of MPS is to generate genomic sequences, but the technology can also be used to create a diagnostic assay with the capacity to detect and potentially strain type any and all pathogens in a sample. This would give MPS diagnostics the capacity to act as a forensic tool, and potentially allow the screening of DNA samples for the hallmarks of genetically engineered harmful toxins/proteins. This project would adapt this technology to pathogen identification by using a "reverse search" tool, to search an MPS database with a limited number of key pathogen sequences. This will cut processing time exponentially, increasing efficiency and throughput. DNA extractions from plants infected with pathogens of interest will be subjected to MPS sequencing. The sequence data will be used to create a database that is "probed" bioinformatically to determine the microbial population present. The technique can also be used for forensic analysis and/or to search for genetically modified microbes by changing the signature sequences used for probing.