USDA-APHIS Plant Germplasm Quarantine Program, Beltsville, MD 20705, USA.
Department of Agriculture, Agribusiness, Environmental Sciences, Texas A&M University-Kingsville, Kingsville, TX 78363, USA.
Viruses. 2021 Aug 17;13(8):1627. doi: 10.3390/v13081627.
Rapid global germplasm trade has increased concern about the spread of plant pathogens and pests across borders that could become established, affecting agriculture and environment systems. Viral pathogens are of particular concern due to their difficulty to control once established. A comprehensive diagnostic platform that accurately detects both known and unknown virus species, as well as unreported variants, is playing a pivotal role across plant germplasm quarantine programs. Here we propose the addition of high-throughput sequencing (HTS) from total RNA to the routine quarantine diagnostic workflow of sugarcane viruses. We evaluated the impact of sequencing depth needed for the HTS-based identification of seven regulated sugarcane RNA/DNA viruses across two different growing seasons (spring and fall). Our HTS analysis revealed that viral normalized read counts (RPKM) was up to 23-times higher in spring than in the fall season for six out of the seven viruses. Random read subsampling analyses suggested that the minimum number of reads required for reliable detection of RNA viruses was 0.5 million, with a viral genome coverage of at least 92%. Using an HTS-based total RNA metagenomics approach, we identified all targeted viruses independent of the time of the year, highlighting that higher sequencing depth is needed for the identification of DNA viruses.
快速的全球种质贸易增加了人们对病原体和害虫跨越边境传播的担忧，这些病原体和害虫可能会在当地定殖，从而影响农业和环境系统。由于病毒病原体一旦定殖就难以控制，因此它们尤其令人关注。一个全面的诊断平台，能够准确检测已知和未知的病毒物种以及未报告的变体，在植物种质检疫计划中发挥着关键作用。在这里，我们建议在甘蔗病毒的常规检疫诊断工作流程中增加来自总 RNA 的高通量测序 (HTS)。我们评估了在两个不同生长季节（春季和秋季）中，基于 HTS 的七种受监管的甘蔗 RNA/DNA 病毒鉴定所需的测序深度的影响。我们的 HTS 分析表明，在七个病毒中有六个病毒的春季病毒归一化读数 (RPKM) 比秋季高 23 倍。随机读取抽样分析表明，可靠检测 RNA 病毒所需的最小读取次数为 50 万次，病毒基因组覆盖率至少为 92%。使用基于 HTS 的总 RNA 宏基因组学方法，我们独立于一年中的时间识别出所有目标病毒，这表明需要更高的测序深度来鉴定 DNA 病毒。
