Department of Fisheries and Wildlife, Michigan State University, East Lansing, MI 48824, USA.
Department of Environmental Health Sciences, School of Public Health and Tropical Medicine, Tulane University, New Orleans, LA 70112, USA.
J Virol Methods. 2020 Apr;278:113805. doi: 10.1016/j.jviromet.2019.113805. Epub 2019 Dec 28.
The newly emerged nanopore sequencing technology such as MinION™ allows for real-time detection of long DNA/RNA fragments on a portable device, yet few have examined its performance for environmental viromes. Here we seeded one RNA virus bacteriophage MS2 and one DNA virus bacteriophage PhiX174 into 10 L well water at three levels ranging from 1 to 21,100 plaque-forming units (PFU)/mL. Two workflows were established to maximize the number of sequencing reads of RNA and DNA viruses using MinION™. With dead-end ultrafiltration, PEG precipitation, and random amplification, MinION™ was capable of detecting MS2 at 155 PFU/mL and PhiX174 at 1-2 PFU/mL. While the DNA workflow only detected PhiX174, the RNA workflow detected both MS2 and PhiX174. The virus concentration, or relative abundance of viral nucleic acids in total nucleic acids, is critical to the proportion of viral reads in sequencing results. Our findings also highlight the importance of including control samples in sequencing runs for environmental water samples with low virus abundance.
新兴的纳米孔测序技术，如 MinION™，允许在便携式设备上实时检测长 DNA/RNA 片段，但很少有人研究其在环境病毒组学中的性能。在这里，我们将一种 RNA 病毒噬菌体 MS2 和一种 DNA 病毒噬菌体 PhiX174 接种到 10 L 井水样本中，浓度范围从 1 到 21100 噬菌斑形成单位 (PFU)/mL。建立了两种工作流程，以最大限度地增加 MinION™ 测序 RNA 和 DNA 病毒的读段数量。通过末端超滤、PEG 沉淀和随机扩增，MinION™ 能够检测到 155 PFU/mL 的 MS2 和 1-2 PFU/mL 的 PhiX174。虽然 DNA 工作流程仅检测到 PhiX174，但 RNA 工作流程同时检测到了 MS2 和 PhiX174。病毒浓度或病毒核酸在总核酸中的相对丰度对测序结果中病毒读段的比例至关重要。我们的研究结果还强调了在测序运行中包含对照样本的重要性，对于病毒丰度低的环境水样尤其如此。
