Department of Plant Pathology, University of California-Davis, Davis, CA 95616, USA.
Foundation Plant Services, University of California-Davis, Davis, CA 95616, USA.
Viruses. 2021 Jun 11;13(6):1130. doi: 10.3390/v13061130.
Development of High-Throughput Sequencing (HTS), also known as next generation sequencing, revolutionized diagnostic research of plant viruses. HTS outperforms bioassays and molecular diagnostic assays that are used to screen domestic and quarantine grapevine materials in data throughput, cost, scalability, and detection of novel and highly variant virus species. However, before HTS-based assays can be routinely used for plant virus diagnostics, performance specifications need to be developed and assessed. In this study, we selected 18 virus-infected grapevines as a test panel for measuring performance characteristics of an HTS-based diagnostic assay. Total nucleic acid (TNA) was extracted from petioles and dormant canes of individual samples and constructed libraries were run on Illumina NextSeq 500 instrument using a 75-bp single-end read platform. Sensitivity was 98% measured over 264 distinct virus and viroid infections with a false discovery rate (FDR) of approximately 1 in 5 positives. The results also showed that combining a spring petiole test with a fall cane test increased sensitivity to 100% for this TNA HTS assay. To evaluate extraction methodology, these results were compared to parallel dsRNA extractions. In addition, in a more detailed dilution study, the TNA HTS assay described here consistently performed well down to a dilution of 5%. In that range, sensitivity was 98% with a corresponding FDR of approximately 1 in 5. Repeatability and reproducibility were assessed at 99% and 93%, respectively. The protocol, criteria, and performance levels described here may help to standardize HTS for quality assurance and accreditation purposes in plant quarantine or certification programs.
高通量测序（HTS），也称为下一代测序，彻底改变了植物病毒的诊断研究。与用于筛选国内和检疫葡萄材料的生物测定和分子诊断测定相比，HTS 在数据吞吐量、成本、可扩展性以及新型和高度变异病毒物种的检测方面表现出色。然而，在 HTS 为基础的检测方法可以常规用于植物病毒诊断之前，需要开发和评估性能规格。在这项研究中，我们选择了 18 株感染病毒的葡萄藤作为测试面板，用于测量基于 HTS 的诊断检测的性能特征。从单个样本的叶柄和休眠藤茎中提取总核酸（TNA），并在 Illumina NextSeq 500 仪器上使用 75bp 单端读取平台运行构建的文库。灵敏度为 98%，在 264 种不同的病毒和类病毒感染中，假阳性率（FDR）约为 1/5。结果还表明，将春季叶柄测试与秋季藤茎测试相结合，可将 TNA HTS 检测的灵敏度提高到 100%。为了评估提取方法，将这些结果与平行的 dsRNA 提取进行了比较。此外，在更详细的稀释研究中，本文描述的 TNA HTS 检测在 5%的稀释度下表现良好。在该范围内，灵敏度为 98%，相应的 FDR 约为 1/5。重复性和再现性分别评估为 99%和 93%。这里描述的方案、标准和性能水平可能有助于为植物检疫或认证计划中的质量保证和认证目的标准化 HTS。
