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Open AccessArticle

A Rapid Method for Sequencing Double-Stranded RNAs Purified from Yeasts and the Identification of a Potent K1 Killer Toxin Isolated from Saccharomyces cerevisiae

1
Department of Biological Sciences, University of Idaho, Moscow, ID 83844, USA
2
IBEST Genomics Core, University of Idaho, Moscow, ID 83843, USA
*
Author to whom correspondence should be addressed.
Viruses 2019, 11(1), 70; https://doi.org/10.3390/v11010070
Received: 31 October 2018 / Revised: 9 January 2019 / Accepted: 11 January 2019 / Published: 16 January 2019
(This article belongs to the Special Issue Mycoviruses)
Mycoviruses infect a large number of diverse fungal species, but considering their prevalence, relatively few high-quality genome sequences have been determined. Many mycoviruses have linear double-stranded RNA genomes, which makes it technically challenging to ascertain their nucleotide sequence using conventional sequencing methods. Different specialist methodologies have been developed for the extraction of double-stranded RNAs from fungi and the subsequent synthesis of cDNAs for cloning and sequencing. However, these methods are often labor-intensive, time-consuming, and can require several days to produce cDNAs from double-stranded RNAs. Here, we describe a comprehensive method for the rapid extraction and sequencing of dsRNAs derived from yeasts, using short-read next generation sequencing. This method optimizes the extraction of high-quality double-stranded RNAs from yeasts and 3′ polyadenylation for the initiation of cDNA synthesis for next-generation sequencing. We have used this method to determine the sequence of two mycoviruses and a double-stranded RNA satellite present within a single strain of the model yeast Saccharomyces cerevisiae. The quality and depth of coverage was sufficient to detect fixed and polymorphic mutations within viral populations extracted from a clonal yeast population. This method was also able to identify two fixed mutations within the alpha-domain of a variant K1 killer toxin encoded on a satellite double-stranded RNA. Relative to the canonical K1 toxin, these newly reported mutations increased the cytotoxicity of the K1 toxin against a specific species of yeast. View Full-Text
Keywords: mycovirus; dsRNA; sequencing; killer toxin; totivirus mycovirus; dsRNA; sequencing; killer toxin; totivirus
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Crabtree, A.M.; Kizer, E.A.; Hunter, S.S.; Van Leuven, J.T.; New, D.D.; Fagnan, M.W.; Rowley, P.A. A Rapid Method for Sequencing Double-Stranded RNAs Purified from Yeasts and the Identification of a Potent K1 Killer Toxin Isolated from Saccharomyces cerevisiae. Viruses 2019, 11, 70.

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