Department of Biological Sciences, University of Idaho, Moscow, ID 83844, USA.
IBEST Genomics Core, University of Idaho, Moscow, ID 83843, USA.
Viruses. 2019 Jan 16;11(1):70. doi: 10.3390/v11010070.
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 . 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.
真菌病毒感染了大量不同的真菌物种，但考虑到它们的普遍性，只有相对较少的高质量基因组序列被确定。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来从真菌中提取双链 RNA，并随后合成 cDNA 进行克隆和测序。然而，这些方法通常劳动强度大、耗时且从双链 RNA 产生 cDNA 可能需要数天时间。在这里，我们描述了一种从酵母中快速提取和测序 dsRNA 的综合方法，使用短读长下一代测序。该方法优化了从酵母中提取高质量双链 RNA 的方法，并对 cDNA 合成进行 3'多聚腺苷酸化，以进行下一代测序。我们已经使用该方法确定了模型酵母 中单个菌株内存在的两种真菌病毒和一种双链 RNA 卫星的序列。所获得的序列质量和覆盖深度足以检测从克隆酵母群体中提取的病毒群体中的固定和多态突变。该方法还能够鉴定卫星双链 RNA 上编码的变体 K1 杀伤毒素的 alpha 结构域内的两个固定突变。与典型的 K1 毒素相比，这些新报告的突变增加了 K1 毒素对特定酵母物种的细胞毒性。
