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J Clin Virol. 2015 Jul;68:43-8. doi: 10.1016/j.jcv.2015.04.019. Epub 2015 Apr 28.

A simplified Sanger sequencing method for full genome sequencing of multiple subtypes of human influenza A viruses.

Author information

1
WHO Collaborating Centre for Reference and Research on Influenza, VIDRL, at the Peter Doherty Institute for Infection and Immunity, Melbourne, Victoria 3000, Australia. Electronic address: yi-mo.deng@influenzacentre.org.
2
WHO Collaborating Centre for Reference and Research on Influenza, VIDRL, at the Peter Doherty Institute for Infection and Immunity, Melbourne, Victoria 3000, Australia.
3
WHO Collaborating Centre for Reference and Research on Influenza, VIDRL, at the Peter Doherty Institute for Infection and Immunity, Melbourne, Victoria 3000, Australia; School of Applied and Biomedical Sciences, Federation University, Churchill, Victoria 3842, Australia.

Abstract

BACKGROUND:

Full genome sequencing of influenza A viruses (IAV), including those that arise from annual influenza epidemics, is undertaken to determine if reassorting has occurred or if other pathogenic traits are present. Traditionally IAV sequencing has been biased toward the major surface glycoproteins haemagglutinin and neuraminidase, while the internal genes are often ignored. Despite the development of next generation sequencing (NGS), many laboratories are still reliant on conventional Sanger sequencing to sequence IAV.

OBJECTIVES:

To develop a minimal and robust set of primers for Sanger sequencing of the full genome of IAV currently circulating in humans.

STUDY DESIGN:

A set of 13 primer pairs was designed that enabled amplification of the six internal genes of multiple human IAV subtypes including the recent avian influenza A(H7N9) virus from China. Specific primers were designed to amplify the HA and NA genes of each IAV subtype of interest. Each of the primers also incorporated a binding site at its 5'-end for either a forward or reverse M13 primer, such that only two M13 primers were required for all subsequent sequencing reactions.

RESULTS:

This minimal set of primers was suitable for sequencing the six internal genes of all currently circulating human seasonal influenza A subtypes as well as the avian A(H7N9) viruses that have infected humans in China.

CONCLUSIONS:

This streamlined Sanger sequencing protocol could be used to generate full genome sequence data more rapidly and easily than existing influenza genome sequencing protocols.

KEYWORDS:

Full genome sequencing; Influenza A virus; Sanger sequencing

PMID:
26071334
DOI:
10.1016/j.jcv.2015.04.019
[Indexed for MEDLINE]
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