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Detection of multiple oomycetes in metagenomic data by Using E-probe Detection of Nucleic Analysis (EDNA)

Maria Proano: Oklahoma State University

<div>Species identification of plant pathogenic oomycetes based on morphology is challenging. Hence, time demanding isolation and ITS sequencing for molecular identification are routinely conducted for diagnostic purposes. Metagenomic diagnostic methods allow identification of multiple pathogens from a single sample without isolation of pure cultures or specific gene-targeted sequencing. E-probe Diagnostic Nucleic acid Analysis (EDNA), a novel method that couples next-generation sequencing and bioinformatics, was used to detect a variety of plant pathogenic oomycetes in metagenomic data. Short pathogen-associated sequences (E-probes), with a range of 60-120 nucleotides, were designed and tested on metadata containing the genomes of a known host and pathogenic oomycetes. E-probes were designed for each pathogen by comparing its genome to the genome of the closest relative available and identifying unique species-specific sequences. To avoid false positives, E-probes with similarity to non-target sequences in NCBI’s Nucleotide database were removed. Mock sequencing databases (MSDs) were created using MetaSim. Resulting databases contained 10’000,000 reads at different titers of pathogen abundances. A metasample was considered positive for the presence of a pathogen when a significant number of e-probes were found in a MSD. EDNA detected the target pathogens in metadata with high accuracy, thus, it is a powerful tool for detection of oomycetes from metasamples.</div>

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