ZOU Xiao Hui; CHEN Wen Bing; ZHAO Xiang; ZHU Wen Fei; YANG Lei; WANG Da Yan; SHU Yue Long

doi:10.3967/bes2016.004

doi: 10.3967/bes2016.004

基金项目: This work was funded by a project (2014ZX10004002) of the ChineseNational Key Program of Mega Infectious Disease of the National 12th Five-Year Plan

Evaluation ofA Single-reaction Method for Whole Genome Sequencing of Influenza A Virus using Next Generation Sequencing

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

Funds: This work was funded by a project (2014ZX10004002) of the ChineseNational Key Program of Mega Infectious Disease of the National 12th Five-Year Plan

摘要: ObjectiveTo 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. MethodsVirus 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. ResultsThe 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. ConclusionThe M-RTPCR is a robust and sensitive amplification method for whole genome sequencing of influenza A virus using NGS platform.
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Abstract: ObjectiveTo 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. MethodsVirus 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. ResultsThe 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. ConclusionThe M-RTPCR is a robust and sensitive amplification method for whole genome sequencing of influenza A virus using NGS platform.
- Influenza A virus /
- Whole genome sequencing /
- NGS

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doi: 10.3967/bes2016.004

Evaluation ofA Single-reaction Method for Whole Genome Sequencing of Influenza A Virus using Next Generation Sequencing

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Evaluation ofA Single-reaction Method for Whole Genome Sequencing of Influenza A Virus using Next Generation Sequencing

doi: 10.3967/bes2016.004

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Evaluation ofA Single-reaction Method for Whole Genome Sequencing of Influenza A Virus using Next Generation Sequencing

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

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doi: 10.3967/bes2016.004

Evaluation ofA Single-reaction Method for Whole Genome Sequencing of Influenza A Virus using Next Generation Sequencing

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Evaluation ofA Single-reaction Method for Whole Genome Sequencing of Influenza A Virus using Next Generation Sequencing

doi: 10.3967/bes2016.004

English Abstract

Evaluation ofA Single-reaction Method for Whole Genome Sequencing of Influenza A Virus using Next Generation Sequencing

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

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