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• Smith, D
• McDonald, J
• Woodward, M
• McCarthy, A
• Allison, H
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• Smith, D
• McDonald, J
• Woodward, M
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• Smith, D
• McDonald, J
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• McCarthy, A
• Allison, H

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Genes 2010, 1(2), 210-226; doi:10.3390/genes1020210

Article

454-Pyrosequencing: A Molecular Battiscope for Freshwater Viral Ecology

David J. Rooks ¹ , Darren L. Smith ^1,† , James E. McDonald ^1,‡ , Martin J. Woodward ² , Alan J. McCarthy ¹  and Heather E. Allison ^1,*

¹ Microbiology Research Group, School of Biological Sciences, Biosciences Building, University of Liverpool, Crown Street, Liverpool, L69 7ZB, UK ² Veterinary Laboratories Agency (Weybridge), New Haw, Addlestone, Surrey KT15 3NB, UK ^† Present address: Liverpool HIV Pharmacology Group, The University of Liverpool, Pembroke Place, Liverpool, L69 3GF, UK. ^‡ Present address: School of Biological Sciences, Bangor University, Deiniol Road, Bangor, Gwynedd, LL57 2UW, UK.

* Author to whom correspondence should be addressed.

Received: 11 May 2010; in revised form: 8 July 2010 / Accepted: 20 July 2010 / Published: 21 July 2010

(This article belongs to the Special Issue Next Generation DNA Sequencing)

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Abstract: Viruses, the most abundant biological entities on the planet, are capable of infecting organisms from all three branches of life, although the majority infect bacteria where the greatest degree of cellular diversity lies. However, the characterization and assessment of viral diversity in natural environments is only beginning to become a possibility. Through the development of a novel technique for the harvest of viral DNA and the application of 454 pyrosequencing, a snapshot of the diversity of the DNA viruses harvested from a standing pond on a cattle farm has been obtained. A high abundance of viral genotypes (785) were present within the virome. The absolute numbers of lambdoid and Shiga toxin (Stx) encoding phages detected suggested that the depth of sequencing had enabled recovery of only ca. 8% of the total virus population, numbers that agreed within less than an order of magnitude with predictions made by rarefaction analysis. The most abundant viral genotypes in the pond were bacteriophages (93.7%). The predominant viral genotypes infecting higher life forms found in association with the farm were pathogens that cause disease in cattle and humans, e.g. members of the Herpesviridae. The techniques and analysis described here provide a fresh approach to the monitoring of viral populations in the aquatic environment, with the potential to become integral to the development of risk analysis tools for monitoring the dissemination of viral agents of animal, plant and human diseases.

Keywords: virome production; viral pathogens; pathogen dissemination; metagenomics; viral ecology

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