Klaudia Chrzastek, Holly S Sellers, Darrell R Kapczynski
{"title":"A Universal, Single-Primer Amplification Protocol to Perform Whole-Genome Sequencing of Segmented dsRNA Avian Orthoreoviruses.","authors":"Klaudia Chrzastek, Holly S Sellers, Darrell R Kapczynski","doi":"10.1637/aviandiseases-D-22-99999","DOIUrl":null,"url":null,"abstract":"<p><p>The <i>Reoviridae</i> 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 <i>Reoviridae</i> 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.</p>","PeriodicalId":8667,"journal":{"name":"Avian Diseases","volume":"66 4","pages":"479-485"},"PeriodicalIF":1.3000,"publicationDate":"2022-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"2","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"Avian Diseases","FirstCategoryId":"97","ListUrlMain":"https://doi.org/10.1637/aviandiseases-D-22-99999","RegionNum":4,"RegionCategory":"农林科学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q2","JCRName":"VETERINARY SCIENCES","Score":null,"Total":0}
引用次数: 2
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.
期刊介绍:
Avian Diseases is an international journal dedicated to publishing original basic or clinical research of the highest quality from various disciplines including microbiology, immunology, pathology and epidemiology. Papers on avian diseases relevant to etiology, pathogenesis, diagnosis, treatment, and control are accepted. Manuscripts dealing with avian species other than poultry will be considered only if the subject is relevant to poultry health.