Comparison of direct RNA sequencing of Orthoavulavirus javaense using two different chemistries on the MinION platform

Authors: Mears, Megan; Read, Quentin; Bakre, Abhijeet

Submitted to: Journal of Virological Methods
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 12/21/2024
Publication Date: N/A
Citation: N/A

Interpretive Summary: Newcastle disease (ND) outbreaks due to virulent ND virus (NDV) can cause significant economic damage to the poultry industry. Owing to its stability in tissues and the environment, NDV can spread rapidly in flocks causing bird death and ensuing monetary loss. This spread can be restricted if NDV is rapidly identified and characterized by sequencing for features that determine virulence. NDV carries RNA as genetic material and traditional methods to differentiate virulent from non-virulent strains rely on amplification which can introduce bias. In this manuscript, we describe a methodology which enables direct sequencing of the viral RNA using a MinION device without amplification. We show that the method enables us to determine the sequence of the full length NDV RNA and thus has potential to be applied in the field. The ability to rapidly identify virus sequences during an outbreak can thus assist in reducing NDV spread and resulting economic loss.

Technical Abstract: Rapidly identifying and sequencing viral pathogens in poultry flocks can substantially reduce economic loss especially during disease outbreaks. Current next generation sequencing technologies require multi-step laboratory-intensive workflows to generate sequence data which precludes field adaptation. In this study, we hypothesized that direct RNA sequencing (DRS) using an Oxford Nanopore Technology (ONT) MinION device would enable sequencing of the full-length viral RNA genome of Orthoavulavirus javaense (OAVJ), the causative of Newcastle disease, a major poultry challenge. The data demonstrate that a custom OAVJ-specific adapter paired with the ONT DRS kits enables capture and sequencing of OAVJ viral RNAs. Further, the new ONT SQK-RNA004 chemistry and flow cells, paired with the associated super accurate base calling workflow improves on read quality and length compared to the previous SQK-RNA002 chemistry. This is the first report of a method to sequence near full-length viral RNA genome of a member of the Paramyxoviridae family. While additional improvements in DRS are needed before widespread adaptation of this method for rapid field sequencing, DRS of OAVJ has the potential to enable further studies into the viral epitranscriptome and its role in infection and pathogenesis.