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

IF 1.3 4区 农林科学 Q2 VETERINARY SCIENCES Avian Diseases Pub Date : 2022-12-01 DOI:10.1637/aviandiseases-D-22-99999
Klaudia Chrzastek, Holly S Sellers, Darrell R Kapczynski
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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.

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一种通用的单引物扩增方法对分段dsRNA禽正肠病毒进行全基因组测序。
呼肠孤病毒科是最大的双链RNA病毒家族,其成员已从广泛的哺乳动物、鸟类、爬行动物、鱼类、昆虫和植物中分离出来。呼肠孤病毒是呼肠孤病毒科15个公认属之一,可感染人类和几乎所有哺乳动物和鸟类。呼肠孤病毒的基因组特征尚未大规模采用,因为获得所有10个片段的序列很复杂。在这项研究中,我们开发了一种高效实用的方法来丰富呼肠孤病毒分离物的测序读数,该方法使用单引物扩增方法与下一代测序相结合,允许全基因组恢复。我们称之为呼肠孤病毒单引物扩增(R-SPA)。我们的研究结果表明,大多数基因在每个碱基空间至少被500个reads覆盖。此外,R-SPA覆盖每个呼肠孤病毒基因的5'和3'端。总之,本研究提出了一种通用的快速扩增方案,可产生足够的双链cDNA,并促进和加快呼肠孤病毒的全基因组测序。
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来源期刊
Avian Diseases
Avian Diseases 农林科学-兽医学
CiteScore
2.40
自引率
7.10%
发文量
80
审稿时长
3 months
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.
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