A Rapid Method for Sequencing Double-Stranded RNAs Purified from Yeasts and the Identification of a Potent K1 Killer Toxin Isolated from Saccharomyces cerevisiae
A Rapid Method for Sequencing Double-Stranded RNAs Purified from Yeasts and the Identification of a Potent K1 Killer Toxin Isolated from Saccharomyces cerevisiae
复制标题
DOI：
10.3390/v11010070
复制
发表时间：
2019-01-01
期刊：
影响因子：
4.7
通讯作者：
Rowley, Paul A.
中科院分区：
文献类型：
--
作者：
Crabtree, Angela M.;Kizer, Emily A.;Rowley, Paul A.
真菌病毒感染大量不同的真菌物种，但考虑到它们的流行，相对较少的高质量的基因组序列已被确定。许多真菌病毒具有线性双链RNA基因组，这使得使用常规测序方法确定其核苷酸序列在技术上具有挑战性。已经开发了不同的专业方法用于从真菌中提取双链RNA以及随后合成用于克隆和测序的cDNA。然而，这些方法通常是劳动密集型的，耗时的，并且可能需要几天才能从双链RNA产生cDNA。在这里，我们描述了一种综合的方法，用于快速提取和测序来自酵母的dsRNA，使用短读的下一代测序。该方法优化了从酵母中提取高质量双链RNA和3聚腺苷酸化，用于启动下一代测序的cDNA合成。我们已经使用这种方法来确定两个真菌病毒和一个双链RNA卫星存在于一个单一的模型酵母酿酒酵母菌株的序列。覆盖的质量和深度足以检测从克隆酵母群体中提取的病毒群体内的固定和多态性突变。该方法还能够鉴定在卫星双链RNA上编码的变体K1杀手毒素的α结构域内的两个固定突变。相对于经典的K1毒素，这些新报道的突变增加了K1毒素对特定酵母菌的细胞毒性。
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.