The Samuel Roberts Noble Foundation, Ardmore, OK 73402, USA.
Mol Ecol. 2010 Mar;19 Suppl 1:81-8. doi: 10.1111/j.1365-294X.2009.04470.x.
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.
在宏基因组研究中对环境样本中的病毒进行了分析，但这些研究并未将单个病毒与其宿主联系起来。我们设计了一种从单个植物中分离双链 RNA 的策略，双链 RNA 是 RNA 病毒感染的标志，并将其转化为 cDNA，每个末端都有独特的四个核苷酸标签。使用 96 个不同的标签允许我们对样本进行混合，同时仍然保留与原始样本的联系。然后，我们使用罗氏 454 焦磷酸测序的大规模平行测序分析混合样本的序列，使得每 picotiter 板可以评估 384 个样本。使用这种方法，我们已经能够分析数千种植物，并且已经发现了数千种新的植物病毒，这些病毒都与其特定的植物宿主相关联。在这里，我们详细描述了该方法，包括对八个样本池的结果和分析。这项技术对于了解植物病毒生物多样性的全貌将非常有用。
