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ARS Home » Southeast Area » Athens, Georgia » U.S. National Poultry Research Center » Endemic Poultry Viral Diseases Research » Research » Publications at this Location » Publication #406594

Research Project: Systems Biology Approaches to Develop Medical Countermeasures to Detect, Prevent, and Control Poultry Production Viral Diseases

Location: Endemic Poultry Viral Diseases Research

Title: Optimizing the conditions for whole-genome sequencing of avian reoviruses

item Alvarez-Narvaez, Sonsiray
item HARRELL, TELVIN - Orise Fellow
item OLUWAYINKA, OLATUNDE - University Of Georgia
item SELLERS, HOLLY - University Of Georgia
item KHALID, ZUBAIR - Auburn University
item HAUCK, RUEDIGER - Auburn University
item CHOWDHURY, ERFAN - Alabama State Governement
item Conrad, Steven

Submitted to: Viruses
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 9/14/2023
Publication Date: 9/16/2023
Citation: Alvarez Narvaez, S., Harrell, T.L., Oluwayinka, O., Sellers, H.S., Khalid, Z., Hauck, R., Chowdhury, E.U., Conrad, S.J. 2023. Optimizing the conditions for whole-genome sequencing of avian reoviruses. Viruses. 15(9):1938.

Interpretive Summary: Avian Reoviruses are a continuing problem for poultry producers and integrators. To better understand them we need to sequence their genomes. This is a challenging task because of the viral genetic concentration and purification requirements. In this paper we describe a new workflow which will allow for the easy and economical sequencing of avian reovirus genomes. We hope that this will provide a gold standard for future avian reovirus sequencing.

Technical Abstract: Whole-genome sequencing (WGS) is becoming an essential tool to characterize the genomes of avian reovirus (ARV), a viral disease of economic significance to poultry producers. The current strategies and procedures used to obtain the complete genome sequences of ARV isolates are not cost-effective because most of the genetic material data resulting from next-generation sequencing belong to the host and cannot be used to assemble the viral genome. The purpose of this study was to develop a workflow to enrich the ARV genomic content in a sample before subjecting it to next-generation sequencing (NGS). Herein, we compare four different ARV purification and enrichment approaches at the virion, RNA and cDNA levels to determine which treatment or treatment combination would provide a higher proportion of ARV-specific reads after WGS. Seven ARV isolates were subjected to different combinations of virion purification via ultracentrifugation in sucrose density gradient or Capto Core 700 resin with or without a subsequent Benzonase treatment, followed by a chicken rRNA depletion step after RNA extraction and a final ARV cDNA amplification step using a single-primer amplification assay. Our results show that the combination of Capto Core 700 resin, Chicken rRNA depletion and cDNA amplification is the most cost-effective strategy to obtain ARV whole genomes after short-read sequencing.