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Next generation sequencing as a tool for pathogen detection in plant introductions grown in quarantine

Martha Malapi-Wight: USDA-APHIS, Plant Germplasm Quarantine Program

<div>The USDA-APHIS Plant Germplasm Quarantine Program (PGQP) is the first line of defense against the entry, establishment, and spread of quarantine-significant pathogens that could harm U.S. agriculture and environment. Plants maintained at PGQP are tested for multiple pathogens using a combination of molecular methods (i.e. PCR and qPCR), serological tests, and bioassays. However, these tests require previous knowledge of the pathogens infecting each crop. Next generation sequencing (NGS) technologies have provided a powerful alternative for detecting and identifying microorganisms without prior knowledge of their presence in that crop. Our goal at PGQP is to establish NGS as a routine diagnostic tool to detect known and novel pathogens in each imported plant accession. In this study, we used Illumina sequencing-by-synthesis technology to sequence >70 quarantined <em>Poaceae</em> clonal accessions that included sugarcanes, turfgrasses, ornamental grasses, and bamboos. Ribosomal-depleted RNA libraries for metagenomics sequencing were generated using the TruSeq Stranded Total RNA with Ribo-Zero Plant Kit and sequenced on an Illumina NextSeq 500 platform. Sequence reads were processed using CLC Genomics Workbench and analyzed using a custom database of 430 viral genomes. A preliminary comparison between conventional laboratory diagnostic tests used by PGQP and NGS technologies suggests that NGS is a powerful tool for diagnostics of pathogens of quarantine importance.</div>