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Viruses. 2019 Jan 16;11(1). pii: E70. doi: 10.3390/v11010070.

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

Author information

1
Department of Biological Sciences, University of Idaho, Moscow, ID 83844, USA. amcrabtree@uidaho.edu.
2
Department of Biological Sciences, University of Idaho, Moscow, ID 83844, USA. mrs@jkizer.com.
3
IBEST Genomics Core, University of Idaho, Moscow, ID 83843, USA. shunter@uidaho.edu.
4
Department of Biological Sciences, University of Idaho, Moscow, ID 83844, USA. jvanleuven@uidaho.edu.
5
IBEST Genomics Core, University of Idaho, Moscow, ID 83843, USA. dnew@uidaho.edu.
6
IBEST Genomics Core, University of Idaho, Moscow, ID 83843, USA. mfagnan@uidaho.edu.
7
Department of Biological Sciences, University of Idaho, Moscow, ID 83844, USA. prowley@uidaho.edu.

Abstract

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.

KEYWORDS:

dsRNA; killer toxin; mycovirus; sequencing; totivirus

PMID:
30654470
PMCID:
PMC6356530
DOI:
10.3390/v11010070
[Indexed for MEDLINE]
Free PMC Article

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