Detection of Norovirus RNA via Nanopore sequencing

To examine whether Nanopore sequencing can be used for detection of Norovirus GII RNA in complex biological samples, a heavily infected human feces sample was analysed using a PromethION sequencer. Norovirus particles were highly resistant to long term incubation at room temperature and to RNase treatment. Since the amount of polyadenylated RNA was insufficient for direct RNA sequencing, libraries were prepared using the PCR-cDNA protocol. Between 10% and 60% of all Nanopore reads corresponded to Norovirus RNA; however, full-length viral sequences were not detected, probably due to strong secondary structures in the Norovirus template RNA that could not be resolved, even when cDNA synthesis was performed at high temperature. In summary, Nanopore sequencing technology can be successfully used to detect Norovirus GII RNA molecules in complex biological samples. PCR amplification of the input material is still necessary, which prevents the detection of full-length Norovirus RNA molecules. It is expected that these limitations will be resolved in the coming years and that Nanopore sequencing will become more cost-effective and competitive with current qPCR-based methods.