Ecogenomics: using massively parallel pyrosequencing to understand virus ecology

Authors


  • MJR is a professor of plant biology studying plant and fungal virus ecology and evolution; PS is a postdoctoral fellow interested in plant virus biodiversity; GBW is currently a postdoctoral fellow developing new methods in high throughput sequencing; JQ is currently a postdoctoral fellow developing bioinformatics tools; JDW and HL are bioinformaticists developing methods for sequence analysis; FC is an ecologist studying microbial biodiversity in Costa Rica; GS is currently a postdoctoral fellow studying plant biochemistry and genetics; BAR is a professor studying nucleic acid biochemistry and analysis methods.

Marilyn J. Roossinck, Fax: 580 224 6692; E-mail: mroossinck@noble.org

Abstract

Environmental samples have been analysed for viruses in metagenomic studies, but these studies have not linked individual viruses to their hosts. We designed a strategy to isolate double-stranded RNA, a hallmark of RNA virus infection, from individual plants and convert this to cDNA with a unique four nucleotide Tag at each end. Using 96 different Tags allowed us to pool samples and still retain the link to the original sample. We then analysed the sequence of pooled samples using massively parallel sequencing with Roche 454 pyrosequencing such that 384 samples could be assessed per picotiter plate. Using this method we have been able to analyse thousands of plants, and we have discovered several thousand new plant viruses, all linked to their specific plant hosts. Here we describe the method in detail, including the results and analysis for eight pools of samples. This technology will be extremely useful in understanding the full scope of plant virus biodiversity.

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