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Updated: Mar 26, 2026

Isolation and Genome Analysis of Single Virions using 'Single Virus Genomics'
Published on: May 26, 2013
Evaluation of A Single-reaction Method for Whole Genome Sequencing of Influenza A Virus using Next Generation
Xiao Hui Zou1, Wen Bing Chen1, Xiang Zhao1
1Chinese National Influenza Center, National Institute for Viral Disease Control and Prevention; China CDC, Key Laboratory for Medical Virology, Ministry of Health, Beijing 102206, China.
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.
Conclusion:
The M-RTPCR is a robust and sensitive amplification method for whole genome sequencing of influenza A virus using NGS platform.
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