title: Evaluation of A Single-reaction Method for Whole Genome Sequencing of Influenza A Virus using Next Generation Sequencing.
abstract: OBJECTIVE
  To evaluate a single-reaction genome ampliﬁcation method, the multisegment reverse transcription-PCR (M-RTPCR), for its sensitivity to full genome sequencing of influenza A virus, and the ability to differentiate mix-subtype virus, using the next generation sequencing (NGS) platform.
  
  
  METHODS
  Virus genome copy was quantified and serially diluted to different titers, followed by amplification with the M-RTPCR method and sequencing on the NGS platform. Furthermore, we manually mixed two subtype viruses to different titer rate and amplified the mixed virus with the M-RTPCR protocol, followed by whole genome sequencing on the NGS platform. We also used clinical samples to test the method performance.
  
  
  RESULTS
  The M-RTPCR method obtained complete genome of testing virus at 125 copies/reaction and determined the virus subtype at titer of 25 copies/reaction. Moreover, the two subtypes in the mixed virus could be discriminated, even though these two virus copies differed by 200-fold using this amplification protocol. The sensitivity of this protocol we detected using virus RNA was also confirmed with clinical samples containing low-titer virus.
  
  
  CONCLUSIONS
  The M-RTPCR is a robust and sensitive amplification method for whole genome sequencing of influenza A virus using NGS platform.
authors.affiliation: Chinese National Influenza Center, National Institute for Viral Disease Control and Prevention; China CDC, Key Laboratory for Medical Virology, Ministry of Health, Beijing 102206, China.
authors.forename: Xiao Hui
authors.initials: XH
authors.lastname: Zou
authors.affiliation: Chinese National Influenza Center, National Institute for Viral Disease Control and Prevention; China CDC, Key Laboratory for Medical Virology, Ministry of Health, Beijing 102206, China.
authors.forename: Wen Bing
authors.initials: WB
authors.lastname: Chen
authors.affiliation: Chinese National Influenza Center, National Institute for Viral Disease Control and Prevention; China CDC, Key Laboratory for Medical Virology, Ministry of Health, Beijing 102206, China.
authors.forename: Xiang
authors.initials: X
authors.lastname: Zhao
authors.affiliation: Chinese National Influenza Center, National Institute for Viral Disease Control and Prevention; China CDC, Key Laboratory for Medical Virology, Ministry of Health, Beijing 102206, China.
authors.forename: Wen Fei
authors.initials: WF
authors.lastname: Zhu
authors.affiliation: Chinese National Influenza Center, National Institute for Viral Disease Control and Prevention; China CDC, Key Laboratory for Medical Virology, Ministry of Health, Beijing 102206, China.
authors.forename: Lei
authors.initials: L
authors.lastname: Yang
authors.affiliation: Chinese National Influenza Center, National Institute for Viral Disease Control and Prevention; China CDC, Key Laboratory for Medical Virology, Ministry of Health, Beijing 102206, China.
authors.forename: Da Yan
authors.initials: da Y
authors.lastname: Wang
authors.affiliation: Chinese National Influenza Center, National Institute for Viral Disease Control and Prevention; China CDC, Key Laboratory for Medical Virology, Ministry of Health, Beijing 102206, China.
authors.forename: Yue Long
authors.initials: YL
authors.lastname: Shu
mesh_terms: D014644:Genetic Variation; D016679:Genome, Viral / Q000235:genetics*; D009980:Influenza A virus / Q000235:genetics*; D012367:RNA, Viral / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*
chemical_list: D012367:RNA, Viral
keywords: Influenza A virus; NGS; Whole genome sequencing