A Universal, Single-Primer Amplification Protocol to Perform Whole-Genome Sequencing of Segmented dsRNA Avian Orthoreoviruses. 2022

Klaudia Chrzastek, and Holly S Sellers, and Darrell R Kapczynski
Exotic and Emerging Avian Diseases Research Unit, Southeast Poultry Research Laboratory, U.S. National Poultry Research Center, Agricultural Research Service, USDA, Athens, Georgia 30605, klaudia.chrzastek@gmail.com.

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.

UI MeSH Term Description Entries
D008322 Mammals Warm-blooded vertebrate animals belonging to the class Mammalia, including all that possess hair and suckle their young. Mammalia,Mammal
D011201 Poultry Diseases Diseases of birds which are raised as a source of meat or eggs for human consumption and are usually found in barnyards, hatcheries, etc. The concept is differentiated from BIRD DISEASES which is for diseases of birds not considered poultry and usually found in zoos, parks, and the wild. Disease, Poultry,Diseases, Poultry,Poultry Disease
D012087 Reoviridae A family of unenveloped RNA viruses with cubic symmetry. The twelve genera include ORTHOREOVIRUS; ORBIVIRUS; COLTIVIRUS; ROTAVIRUS; Aquareovirus, Cypovirus, Phytoreovirus, Fijivirus, Seadornavirus, Idnoreovirus, Mycoreovirus, and Oryzavirus. Aquareovirus,Cypovirus,Cytoplasmic Polyhedrosis Viruses,Fijivirus,Idnoreovirus,Mycoreovirus,Oryzavirus,Phytoreovirus,Reoviruses, Aquatic,Respiratory Enteric Orphan Viruses,Seadornavirus,Aquareoviruses,Aquatic Reovirus,Aquatic Reoviruses,Cypoviruses,Cytoplasmic Polyhedrosis Virus,Fijiviruses,Idnoreoviruses,Mycoreoviruses,Oryzaviruses,Phytoreoviruses,Polyhedrosis Virus, Cytoplasmic,Polyhedrosis Viruses, Cytoplasmic,Reovirus, Aquatic,Seadornaviruses
D006801 Humans Members of the species Homo sapiens. Homo sapiens,Man (Taxonomy),Human,Man, Modern,Modern Man
D000818 Animals Unicellular or multicellular, heterotrophic organisms, that have sensation and the power of voluntary movement. Under the older five kingdom paradigm, Animalia was one of the kingdoms. Under the modern three domain model, Animalia represents one of the many groups in the domain EUKARYOTA. Animal,Metazoa,Animalia
D012330 RNA, Double-Stranded RNA consisting of two strands as opposed to the more prevalent single-stranded RNA. Most of the double-stranded segments are formed from transcription of DNA by intramolecular base-pairing of inverted complementary sequences separated by a single-stranded loop. Some double-stranded segments of RNA are normal in all organisms. Double-Stranded RNA,Double Stranded RNA,RNA, Double Stranded
D016679 Genome, Viral The complete genetic complement contained in a DNA or RNA molecule in a virus. Viral Genome,Genomes, Viral,Viral Genomes
D018165 Orthoreovirus A genus of the family REOVIRIDAE infecting vertebrates only. Transmission is horizontal and infected species include humans, birds, cattle, monkeys, sheep, swine, baboons, and bats. MAMMALIAN ORTHOREOVIRUS is the type species. Baboon orthoreovirus,Nelson Bay orthoreovirus,Orthoreovirus, Baboon,Orthoreovirus, Nelson Bay,Orthoreoviruses
D030101 Orthoreovirus, Avian A species of ORTHOREOVIRUS infecting birds, with outcomes ranging from inapparent to lethal depending on the virus strain and age of the host bird. This species does not infect mammals. Avian Reoviruses,Avian orthoreovirus,Avian Reovirus,Reovirus, Avian,Reoviruses, Avian

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