title: Partial purification of IBV and subsequent isolation of viral RNA for next-generation sequencing.
abstract: 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.
authors.affiliation: The Pirbright Institute, High Street, Compton, Newbury, Berkshire, RG20 7NN, UK, sarah.keep@pirbright.ac.uk.
authors.forename: Sarah M
authors.initials: SM
authors.lastname: Keep
authors.forename: Erica
authors.initials: E
authors.lastname: Bickerton
authors.forename: Paul
authors.initials: P
authors.lastname: Britton
mesh_terms: D000818:Animals; D002642:Chick Embryo; D059014:High-Throughput Nucleotide Sequencing; D001351:Infectious bronchitis virus / Q000235:genetics / Q000302:isolation & purification*; D012367:RNA, Viral / Q000235:genetics / Q000302:isolation & purification*; D017423:Sequence Analysis, RNA
chemical_list: D012367:RNA, Viral