Nanopore sequencing for rapid diagnostics of salmonid RNA viruses

Abstract

Analysis of pathogen genome variation is essential for informing disease management and control measures in farmed animals. For farmed fish, the standard approach is to use PCR and Sanger sequencing to study partial regions of pathogen genomes, with second and third-generation sequencing tools yet to be widely applied. Here we demonstrate rapid and accurate sequencing of two disease-causing viruses affecting global salmonid aquaculture, salmonid alphavirus (SAV) and infectious salmon anaemia virus (ISAV), using third-generation nanopore sequencing on the MinION platform (Oxford Nanopore Technologies). Our approach complements PCR from infected material with MinION sequencing to recover genomic information that matches near perfectly to Sanger-verified references. We use this method to present the first SAV subtype-6 genome, which branches as the sister to all other SAV lineages in a genome-wide phylogenetic reconstruction. MinION sequencing offers an effective strategy for fast, genome-wide analysis of fish viruses, with major potential applications for diagnostics and robust investigations into the origins and spread of disease outbreaks.

Genome Sequencing of SAV3 Reveals Repeated Seeding Events of Viral Strains in Norwegian Aquaculture

“…We sequenced 24 SAV3-infected fish hearts from Norway using an overlapping PCR amplicon approach and the MinION Nanopore platform (Table 1). This resulted in an average coverage of 6,459x across samples (minimum average coverage of 1,089x and maximum average coverage of 12,434x); significantly more than the minimum requirement for high consensus sequence accuracy (Gallagher et al, 2018). Of the 24 samples sequenced, near full genomes were recovered from 21 samples (approximately 11,600 bp in length), while partial genomes were recovered from 3 samples (∼ 9,500 bp).…”

“…Therefore, to characterize the current phylogenetic structure of SAV3 and thus test the hypothesis that passive drift by water currents has been the dominating transmission mechanism, an established Nanopore amplicon sequencing approach (Gallagher et al, 2018) was used in the current study. After first confirming the accuracy of this approach for distinguishing sub-type level co-infections of SAV2 and SAV3, which we considered a hypothetical possibility in regions with overlapping epidemics, twenty-four SAV3 genomes were sequenced from heart tissues sampled between 2016 and 2019.…”

Current Trends in Diagnostics of Viral Infections of Unknown Etiology

“…To summarize, small physical dimensions of MinION, along with other ONT sequencers, availability of DNA and RNA sequencing protocols, potentially high precision and high data processing rates create promising prospects for its clinical application [179]. So far, it has already been tested in veterinary science for detection of canine distemper virus (CDV) [203,204] and in biology for identification of numerous pathogens in plants [205] and fish [206]. Nevertheless, prior to extrapolating these positive results into the medical field, we need to develop and validate the workflows that would easily integrate into the everyday work of a clinical laboratory and not require any special bioinformatic training in the lab staff [207].…”

Rapid, Unbiased PRRSV Strain Detection Using MinION Direct RNA Sequencing and Bioinformatics Tools

“…The Oxford Nanopore MinION sequencer allows for sequencing of the RNA molecule directly, in its native format. This feature, together with other characteristics such as low start-up costs, portability, and real-time data streaming, makes the Oxford Nanopore MinION sequencer a good candidate for rapid RNA virus detection, even in resource-limited or remote areas [40,42,43]. Influenza virus was the first pathogen to be successfully sequenced in its native RNA format by direct RNA sequencing (DRS) using Oxford Nanopore MinION technology [44].…”