Partial Purification of IBV and Subsequent Isolation of Viral RNA for Next-Generation Sequencing

Keep, Sarah M.; Bickerton, Erica; Britton, Paul

RNA viruses are known for a high mutation rate and rapid genomic evolution. As such an RNA virus population does not consist of a single genotype but is rather a collection of individual viruses with closely related genotypes—a quasispecies, which can be analyzed by next-generation sequencing (NGS). This diversity of genotypes provides a mechanism in which a virus population can evolve and adapt to a changing environment. Sample preparation is vital for successful sequencing. The following protocol describes the process of generating a high-quality RNA preparation from IBV grown in embryonated eggs and then partially purified and concentrated through a 30 % sucrose cushion for NGS.