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Open Access Highly Accessed Methodology article

Viral genome sequencing by random priming methods

Appolinaire Djikeng1*, Rebecca Halpin1, Ryan Kuzmickas1, Jay DePasse5, Jeremy Feldblyum1, Naomi Sengamalay1, Claudio Afonso2, Xinsheng Zhang3, Norman G Anderson4, Elodie Ghedin5 and David J Spiro1*

Author affiliations

1 Viral Genomics Group, J. Craig Venter Institute, Rockville, MD 20850, USA

2 Southeast Poultry Research Laboratory, Agricultural Research Service, US Department of Agriculture, Athens, GA 30605, USA

3 Food Animal Health Research Program, Ohio Agricultural Research and Development Center, The Ohio State University, Wooster, OH 44691, USA

4 Viral Defense Foundation, Kensington, MD 20891, USA

5 Division of Infectious Diseases, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261, USA

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Citation and License

BMC Genomics 2008, 9:5  doi:10.1186/1471-2164-9-5

Published: 7 January 2008

Abstract

Background

Most emerging health threats are of zoonotic origin. For the overwhelming majority, their causative agents are RNA viruses which include but are not limited to HIV, Influenza, SARS, Ebola, Dengue, and Hantavirus. Of increasing importance therefore is a better understanding of global viral diversity to enable better surveillance and prediction of pandemic threats; this will require rapid and flexible methods for complete viral genome sequencing.

Results

We have adapted the SISPA methodology [1-3] to genome sequencing of RNA and DNA viruses. We have demonstrated the utility of the method on various types and sources of viruses, obtaining near complete genome sequence of viruses ranging in size from 3,000–15,000 kb with a median depth of coverage of 14.33. We used this technique to generate full viral genome sequence in the presence of host contaminants, using viral preparations from cell culture supernatant, allantoic fluid and fecal matter.

Conclusion

The method described is of great utility in generating whole genome assemblies for viruses with little or no available sequence information, viruses from greatly divergent families, previously uncharacterized viruses, or to more fully describe mixed viral infections.