Dataset Information

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.

SUBMITTER: Gallagher MD

PROVIDER: S-EPMC6218516 | biostudies-literature | 2018 Nov

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Nanopore sequencing for rapid diagnostics of salmonid RNA viruses.

Gallagher Michael D MD Matejusova Iveta I Nguyen Lien L Ruane Neil M NM Falk Knut K Macqueen Daniel J DJ

Scientific reports 20181105 1

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 vir ...[more]

PMID: 30397226

Similar Datasets

Project description:RNA viruses are the etiological agents of many infectious diseases. Since RNA viruses are error-prone during genome replication, rapid, accurate and economical whole RNA viral genome sequence determination is highly demanded. Next-generation sequencing (NGS) techniques perform whole viral genome sequencing due to their high-throughput sequencing capacity. However, the NGS techniques involve a significant burden for sample preparation. Since to generate complete viral genome coverage, genomic nucleic acid enrichment is required by reverse transcription PCR using virus-specific primers or by viral particle concentration. Furthermore, conventional NGS techniques cannot determine the 5' and 3' terminal sequences of the RNA viral genome. Therefore, the terminal sequences are determined one by one using rapid amplification of cDNA ends (RACE). However, since some RNA viruses have segmented genomes, the burden of the determination using RACE is proportional to the number of segments. To date, there is only one study attempting whole genome sequencing of multiple RNA viruses without using above mentioned methods, but the generated sequences' accuracy compared to the reference sequences was up to 97% and did not reach 100% due to the low read depth. Hence, we established novel methods, named PCR-NGS and RCA-NGS, that were optimized for an NGS machine, MinION. These methods do not require nucleic acid amplification with virus-specific PCR primers, physical viral particle enrichment, and RACE. These methods enable whole RNA viral genome sequencing by combining the following techniques: (1) removal of unwanted DNA and RNA other than the RNA viral genome by nuclease treatment; (2) the terminal of viral genome sequence determination by barcoded linkers ligation; (3) amplification of the viral genomic cDNA using ligated linker sequences-specific PCR or an isothermal DNA amplification technique, such as rolling circle amplification (RCA). The established method was evaluated using isolated RNA viruses with single-stranded, double-stranded, positive-stranded, negative-stranded, non-segmented or multi-segmented genomes. As a result, all the viral genome sequences could be determined with 100% accuracy, and these mean read depths were greater than 2,500×, at least using either of the methods. This method should allow for easy and economical determination of accurate RNA viral genomes.

Dataset Information

Nanopore sequencing for rapid diagnostics of salmonid RNA viruses.

Publications

Nanopore sequencing for rapid diagnostics of salmonid RNA viruses.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets