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POSTERS: Pathogen detection, quantification and diagnosis

Detecting viruses and bacteria of grapevine with Microbe Finder (MiFi) and an Oxford Nanopore sequencer
Andres Espindola - Oklahoma State University. Kitty Cardwell- Oklahoma State University, Francisco Ochoa Corona- Oklahoma State University, Luís Fernando Maranho Watanabe- Oklahoma State University, Viviana Freire-Zapata- Oklahoma State University

Grapevines can be infected by numerous viruses and bacteria. Infections may be mixed complicating detection and diagnosis. Simultaneous detection of multiple viruses has been performed using High Throughput Sequencing (HTS) and MiFi virus e-probes. Portable sequencers like the minION from Oxford Nanopore Technologies (ONT) allow fast, inexpensive access to HTS. We have developed a graphical-user interphase (GUI), MiFi, that contains e-probe libraries that will allow unequivocal detection of 12 pathogens of grapevine in metagenomic sequences. E-probes are unique signatures of each pathogen of interest used for a targeted query if the pathogen sequence is found in HTS data. Currently there are specific e-probes for bacteria Xylella fastidiosa subs. fastidiosa (14,236) and for Agrobacterium vitis (4,034), and 19 viruses including strains of Grapevine Leafroll associated virus (GLRaV). Sensitivity was assessed in silico where relative gradient of pathogen abundance was simulated using host and pathogen genomes from NCBI. Bacterial pathogens had higher sensitivity (< 0.001%) than viruses (average 0.02%). Positive control samples were sequenced with the minION and sensitivity was assessed using different viral concentrations. Validation with real sequence data (in vitro) has been done for GLRaV-2, GLRaV-3 and TRSV. Preliminary results have shown that MiFi is able to detect the pathogens in minION metagenomic sequence data.