Evaluation of a portable nanopore-based sequencer for detection of viruses in water

•We developed 2 workflows to target DNA and RNA viruses separately using MinION™.•RNA virus MS2 was detected at 155 plaque-forming units/mL in seeded well water.•DNA virus PhiX174 was detected at 1–2 plaque-forming units/mL in seeded well water.•DNA workflow had better sensitivity and selectivity than RNA workflow.•Control samples are a must for environmental samples with low virus abundance. 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.