WSL Swiss Federal Research Institute, Zürcherstrasse 111, 8903 Birmensdorf, Switzerland.
Viruses. 2020 Jul 25;12(8):801. doi: 10.3390/v12080801.
The MinION sequencer is increasingly being used for the detection and outbreak surveillance of pathogens due to its rapid throughput. For RNA viruses, MinION's new direct RNA sequencing is the next significant development. Direct RNA sequencing studies are currently limited and comparisons of its diagnostic performance relative to different DNA sequencing approaches are lacking as a result. We sought to address this gap and sequenced six subtypes from the mycovirus CHV-1 using MinION's direct RNA sequencing and DNA sequencing based on a targeted viral amplicon. Reads from both techniques could correctly identify viral presence and species using BLAST, though direct RNA reads were more frequently misassigned to closely related CHV species. De novo consensus sequences were error prone but suitable for viral species identification. However, subtype identification was less accurate from both reads and consensus sequences. This is due to the high sequencing error rate and the limited sequence divergence between some CHV-1 subtypes. Importantly, neither RNA nor amplicon sequencing reads could be used to obtain reliable intra-host variants. Overall, both sequencing techniques were suitable for virus detection, though limitations are present due to the error rate of MinION reads.
由于其高通量，MinION 测序仪越来越多地用于病原体的检测和爆发监测。对于 RNA 病毒，MinION 的新直接 RNA 测序是下一个重要的发展。直接 RNA 测序研究目前受到限制，并且由于缺乏针对不同 DNA 测序方法的诊断性能比较。我们试图解决这一差距，并使用 MinION 的直接 RNA 测序和基于靶向病毒扩增子的 DNA 测序对真菌病毒 CHV-1 的六个亚型进行测序。使用 BLAST，两种技术的读取都可以正确识别病毒的存在和种类，尽管直接 RNA 读取更频繁地被错误地分配给密切相关的 CHV 物种。从头开始的共识序列容易出错，但适合病毒种类鉴定。然而，无论是读取还是共识序列，亚型鉴定都不够准确。这是由于高测序错误率和一些 CHV-1 亚型之间有限的序列差异。重要的是，RNA 或扩增子测序读取都不能用于获得可靠的宿主内变体。总的来说，两种测序技术都适用于病毒检测，但由于 MinION 读取的错误率，存在一些限制。
