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United States Department of Agriculture

Agricultural Research Service

Title: Viral genome sequencing bt random priming methods)

Author
item Djikeng, A
item Halpin, R
item Kuzmickas, R
item Sengamalay, N
item Afonso, Claudio
item Xang, X
item Anderson, N
item Ghedin, E
item Spiro, D

Submitted to: Biomed Central (BMC) Genomics
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 1/8/2008
Publication Date: 1/30/2008
Citation: Djikeng, A., Halpin, R., Kuzmickas, R., Sengamalay, N., Afonso, C.L., Xang, X., Anderson, N.G., Ghedin, E., Spiro, D.J. 2008. Viral genome sequencing by random priming methods. Biomed Central (BMC) Genomics. 9:5.

Interpretive Summary: The causative agents of many important diseases are viruses. Of increasing importance is an understanding of the viral diversity to enable better surveillance and prediction of pandemic threats. Increased understanding of viral diversity will require rapid and flexible methods for complete genome sequencing. Here a technology for rapid sequencing has been adapted for the complete genome sequencing of several RNA and DNA viruses. obtaining near complete genome sequence of viruses ranging in size from 3000-15,000 kb with a median depth of coverage of 14.33. The utility of this technique to generate full viral genome sequence in the presence of host contaminants, using cell culture purified viruses and viral preps from cell culture supernatant, allantoic fluid and fecal matter is demonstrated. The method described is potentially of great utility in generating whole genome assemblies for viruses with little or no available sequence information, viruses greatly divergent from sequenced family members, and previously uncharacterized viruses.

Technical Abstract: Most emerging health threats are of zoonotic origin. For the overwhelming majority, their causative agents are viruses which include but are not limited to HIV, Influenza, SARS, Ebola, Dengue, and Hantavirus. Of increasing importance therefore is an understanding of the viral diversity to enable better surveillance and prediction of pandemic threats. Increased understanding of viral diversity will require rapid and flexible methods for complete genome sequencing. Results: We have adapted the SISPA-DNase I methodology (Allander et al. 12891-96) to complete 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 3000-15,000 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 cell culture purified viruses and viral preps from cell culture supernatant, allantoic fluid and fecal matter. Conclusion: The method described is potentially of great utility in generating whole genome assemblies for viruses with little or no available sequence information, viruses greatly divergent from sequenced family members, and previously uncharacterized viruses.

Last Modified: 8/24/2016
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