Randomly primed, strand-switching, MinION-based sequencing for the detection and characterization of cultured RNA viruses

Abstract

RNA viruses rapidly mutate, which can result in increased virulence, increased escape from vaccine protection, and false-negative detection results. Targeted detection methods have a limited ability to detect unknown viruses and often provide insufficient data to detect coinfections or identify antigenic variants. Random, deep sequencing is a method that can more fully detect and characterize RNA viruses and is often coupled with molecular techniques or culture methods for viral enrichment. We tested viral culture coupled with third-generation sequencing for the ability to detect and characterize RNA viruses. Cultures of bovine viral diarrhea virus, canine distemper virus (CDV), epizootic hemorrhagic disease virus, infectious bronchitis virus, 2 influenza A viruses, and porcine respiratory and reproductive syndrome virus were sequenced on the MinION platform using a random, reverse primer in a strand-switching reaction, coupled with PCR-based barcoding. Reads were taxonomically classified and used for reference-based sequence building using a stock personal computer. This method accurately detected and identified complete coding sequence genomes with a minimum of 20× coverage depth for all 7 viruses, including a sample containing 2 viruses. Each lineage-typing region had at least 26× coverage depth for all viruses. Furthermore, analyzing the CDV sample through a pipeline devoid of CDV reference sequences modeled the ability of this protocol to detect unknown viruses. Our results show the ability of this technique to detect and characterize dsRNA, negative- and positive-sense ssRNA, and nonsegmented and segmented RNA viruses.

Retrospective Detection and Complete Genomic Sequencing of Canine morbillivirus in Eurasian Otter (Lutra lutra) Using Nanopore Technology

“…We emphasize the importance of providing novel sequence data in accordance with recent studies, in which the complete genomic sequence data of CDV are increasingly reported. This will greatly facilitate a better understanding of both the evolution of CDV and epizootic patterns in the near future [ 47 , 64 , 65 , 66 , 67 ]. In our study, we used a recently published pan-genotype CDV-specific amplicon-based sequencing method developed for the Oxford Nanopore Technologies platform [ 51 ].…”

Detection and sequence analysis of Canine morbillivirus in multiple species of the Mustelidae family

“…Next generation sequencing (NGS) technologies are increasingly being used to detect and characterize pathogens in wildlife [ 41 – 44 ]. MinION (Oxford Nanopore Technologies, Oxford, UK) has been used in many areas of virology, for instance, metagenomics or sequencing of complete genomes [ 45 – 48 ]. Amplicon-based NGS sequencing of specific pathogens is a method for rapid detection and genomic characterization of target pathogens which may yield high-coverage genomic sequence information [ 12 , 49 – 52 ].…”

Discovery of Avian Paramyxoviruses APMV-1 and APMV-6 in Shorebirds and Waterfowl in Southern Ukraine

“…Inadequate sequencing at the beginning and end of the genome in the noncoding region was consistent when sequencing other RNA viruses, such as Zika, using the tiling primer approach [ 29 ]. Newer, randomly primed, strand-switching amplification protocols have been recently developed that may complement tiling primer-based sequencing, with particular strength in the agnostic sequencing of divergent RNA viruses [ 50 ]. However, this protocol was not available at the time of sequencing in Ukraine, and remain to be robustly tested in low-resourced laboratories or field settings.…”