Determining the Suitability of MinION's Direct RNA and DNA Amplicon Sequencing for Viral Subtype Identification.

Abstract

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.