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

Abstract The newly emerged nanopore sequencing technology such as MinIONTM 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 21100 plaque-forming units (PFU)/mL. Two pipelines were established to maximize the number of sequencing reads of DNA and RNA viruses using MinIONTM. With dead-end ultrafiltration, PEG precipitation, and random amplification, MinIONTM was capable of detecting MS2 at 155 PFU/mL and PhiX174 at 1 – 2 PFU/mL. Overall, the DNA pipeline showed higher sensitivity and specificity to PhiX174 than those of the RNA pipeline to MS2. 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.