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ARS Home » Southeast Area » Athens, Georgia » U.S. National Poultry Research Center » Exotic & Emerging Avian Viral Diseases Research » Research » Publications at this Location » Publication #389673
Research Project: Control Strategies to Prevent and Respond to Diseases Outbreaks Caused by Avian Influenza Viruses

Location: Exotic & Emerging Avian Viral Diseases Research

Title: A universal, single-primer amplification protocol to perform whole-genome sequencing of segmented dsRNA avian orthoreoviruses

Author
item CHRZASTEK, KLAUDIA - Orise Fellow
item SELLERS, HOLLY - University Of Georgia
item Kapczynski, Darrell

Submitted to: Avian Diseases
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 7/7/2022
Publication Date: 1/16/2023
Citation: Chrzastek, K., Sellers, H.S., Kapczynski, D.R. 2023. A universal, single-primer amplification protocol to perform whole-genome sequencing of segmented dsRNA avian orthoreoviruses. Avian Diseases. 66(4):479-485. https://doi.org/10.1637/aviandiseases-D-22-99999.
DOI: https://doi.org/10.1637/aviandiseases-D-22-99999

Interpretive Summary: The flexibility of next-generation sequencing (NGS) technology allows almost any genetic material to be studied on a genome-wide scale. Complete and accurate genome sequencing are essential to facilitate full genome assemblies. Genetic characterization of RNA viruses using sequence-based methods is widely used to understand virus diversity, virus spread, understand origin and evolutionary history of viruses or to perform clinical diagnostics. In this study, we developed a simple template enrichment protocol that utilizes one universal primer to target all ten segments of the reovirus genome. This research will expand knowledge of these RNA viruses through examination of their complete genome.

Technical Abstract: The Reoviridae family represents the largest family of double-stranded RNA viruses, and members have been isolated from a wide range of mammals, birds, reptiles, fishes, insects, and plants. Orthoreoviruses, one of the 15 recognized genera in the Reoviridae family, can infect humans and nearly all mammals and birds. Genomic characterization of reoviruses has not been adopted on a large scale because of the complexity of obtaining sequences for all 10 segments. In this study, we develop a time-efficient and practical method to enrich reovirus sequencing reads from isolates that allows for full-genome recovery using a single-primer amplification method coupled with next-generation sequencing. We refer to this protocol as reovirus–single-primer amplification (R-SPA). Our results demonstrate that most of the genes are covered with at least 500 reads per base space. Furthermore, R-SPA covers both the 5' and 3' ends of each reovirus genes. In summary, this study presents a universal and fast amplification protocol that yields sufficient double-stranded cDNA and facilitates and expedites the whole-genome sequencing of reoviruses.