Table_1_NanoViromics: long-read sequencing of dsRNA for plant virus and viroid rapid detection.XLSX

There is a global need for identifying viral pathogens, as well as for providing certified clean plant materials, in order to limit the spread of viral diseases. A key component of management programs for viral-like diseases is having a diagnostic tool that is quick, reliable, inexpensive, and easy to use. We have developed and validated a dsRNA-based nanopore sequencing protocol as a reliable method for detecting viruses and viroids in grapevines. We compared our method, which we term direct-cDNA sequencing from dsRNA (dsRNAcD), to direct RNA sequencing from rRNA-depleted total RNA (rdTotalRNA), and found that it provided more viral reads from infected samples. Indeed, dsRNAcD was able to detect all of the viruses and viroids detected using Illumina MiSeq sequencing (dsRNA-MiSeq). Furthermore, dsRNAcD sequencing was also able to detect low-abundance viruses that rdTotalRNA sequencing failed to detect. Additionally, rdTotalRNA sequencing resulted in a false-positive viroid identification due to the misannotation of a host-driven read. Two taxonomic classification workflows, DIAMOND & MEGAN (DIA & MEG) and Centrifuge & Recentrifuge (Cent & Rec), were also evaluated for quick and accurate read classification. Although the results from both workflows were similar, we identified pros and cons for both workflows. Our study shows that dsRNAcD sequencing and the proposed data analysis workflows are suitable for consistent detection of viruses and viroids, particularly in grapevines where mixed viral infections are common.

当前全球亟需开展病毒病原体识别工作，并获取经认证的清洁植物材料，以遏制病毒病害的传播。针对类病毒病害的防控方案，其核心组成要素之一是具备快速可靠、成本低廉且操作简便特性的诊断工具。我们开发并验证了一种基于双链RNA（dsRNA）的纳米孔测序方案，以此作为检测葡萄植株中病毒与类病毒的可靠手段。我们将该方法命名为基于双链RNA的直接cDNA测序（dsRNAcD），并将其与针对去除核糖体RNA总RNA的直接RNA测序（rdTotalRNA）进行了对比，结果显示，该方法从感染样本中获取的病毒读段数量更多。事实上，dsRNAcD能够检测出所有通过Illumina MiSeq测序（dsRNA-MiSeq）鉴定得到的病毒与类病毒。此外，dsRNAcD测序还可检测到rdTotalRNA测序未能检出的低丰度病毒。同时，rdTotalRNA测序因对宿主来源读段的注释错误，出现了类病毒假阳性鉴定的结果。我们还评估了两套分类学分析流程——DIAMOND与MEGAN（DIA & MEG）以及Centrifuge与Recentrifuge（Cent & Rec），以实现快速且准确的读段分类。尽管两套流程的分析结果相近，但我们仍分别明确了二者的优缺点。本研究表明，dsRNAcD测序与所提出的数据分析流程，可用于稳定检测病毒与类病毒，尤其适用于混合病毒感染普遍存在的葡萄植株样本。