title: Rapid whole genome sequencing of Miyazaki-Bali/2007 Pteropine orthoreovirus by modified rolling circular amplification with adaptor ligation - next generation sequencing.
abstract: The emergence of orthoreoviruses as the causative agent of human respiratory illness over the past few years has led to a demand to determine their viral genome sequences. The whole genome sequencing of such RNA viruses using traditional methods, such as Sanger dideoxy sequencing following rapid amplification of cDNA ends presents a laborious challenge due to the numerous preparatory steps required before sequencing can commence. We developed a practical, time-efficient novel combination method capable of reducing the total time required from months to less than a week in the determination of whole genome sequence of Pteropine orthoreoviruses (PRV); through a combination of viral RNA purification and enrichment, adaptor ligation, reverse transcription, cDNA circularization and amplification, and next generation sequencing. We propose to call the method "modified rolling circular amplification with adaptor ligation - next generation sequencing (mRCA-NGS)". Here, we describe the technological focus and advantage of mRCA-NGS and its expansive application, exemplified through the phylogenetic understanding of the Miyazaki-Bali/2007 PRV.
authors.affiliation: Department of Intelligent Mechanical Systems, Graduate School of Systems Design, Tokyo Metropolitan University, Tokyo, 192-0065, Japan.|Special Pathogens Laboratory, Department of Virology 1, National Institute of Infectious Diseases, Tokyo, 208-0011, Japan.
authors.forename: Harpal
authors.initials: H
authors.lastname: Singh
authors.affiliation: Special Pathogens Laboratory, Department of Virology 1, National Institute of Infectious Diseases, Tokyo, 208-0011, Japan.
authors.forename: Tomoki
authors.initials: T
authors.lastname: Yoshikawa
authors.affiliation: Laboratory of Viral Replication, International Research Center for Infectious Diseases, Research Institute for Microbial Diseases, Osaka University, Osaka, 565-0871, Japan.
authors.forename: Takeshi
authors.initials: T
authors.lastname: Kobayashi
authors.affiliation: Special Pathogens Laboratory, Department of Virology 1, National Institute of Infectious Diseases, Tokyo, 208-0011, Japan.
authors.forename: Shuetsu
authors.initials: S
authors.lastname: Fukushi
authors.affiliation: Special Pathogens Laboratory, Department of Virology 1, National Institute of Infectious Diseases, Tokyo, 208-0011, Japan.
authors.forename: Hideki
authors.initials: H
authors.lastname: Tani
authors.affiliation: Special Pathogens Laboratory, Department of Virology 1, National Institute of Infectious Diseases, Tokyo, 208-0011, Japan.
authors.forename: Satoshi
authors.initials: S
authors.lastname: Taniguchi
authors.affiliation: Special Pathogens Laboratory, Department of Virology 1, National Institute of Infectious Diseases, Tokyo, 208-0011, Japan.
authors.forename: Aiko
authors.initials: A
authors.lastname: Fukuma
authors.affiliation: Department of Intelligent Mechanical Systems, Graduate School of Systems Design, Tokyo Metropolitan University, Tokyo, 192-0065, Japan.
authors.forename: Ming
authors.initials: M
authors.lastname: Yang
authors.affiliation: Department of Hygiene and Public Health, Graduate School of Human Health Sciences, Tokyo Metropolitan University, Tokyo, 192-0397, Japan.
authors.forename: Masami
authors.initials: M
authors.lastname: Sugamata
authors.affiliation: Special Pathogens Laboratory, Department of Virology 1, National Institute of Infectious Diseases, Tokyo, 208-0011, Japan.
authors.forename: Masayuki
authors.initials: M
authors.lastname: Shimojima
authors.affiliation: Special Pathogens Laboratory, Department of Virology 1, National Institute of Infectious Diseases, Tokyo, 208-0011, Japan.
authors.forename: Masayuki
authors.initials: M
authors.lastname: Saijo
mesh_terms: D000818:Animals; D002460:Cell Line; D016679:Genome, Viral; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D006801:Humans; D018165:Orthoreovirus / Q000145:classification / Q000235:genetics* / Q000302:isolation & purification; D010802:Phylogeny; D012367:RNA, Viral / Q000235:genetics; D017422:Sequence Analysis, DNA / Q000379:methods
chemical_list: D012367:RNA, Viral