Bigelow Laboratory for Ocean Sciences, East Boothbay, ME, USA.
ISME J. 2014 May;8(5):1079-88. doi: 10.1038/ismej.2013.214. Epub 2013 Dec 5.
Metagenomics has opened new windows on investigating viral diversity and functions. Viromic studies typically require large sample volumes and filtration through 0.2 μm pore-size filters, consequently excluding or under-sampling tailed and very large viruses. We have optimized a targeted viromic approach that employs fluorescence-activated sorting and whole genome amplification to produce dsDNA-enriched libraries from discrete viral populations from a 1-ml water sample. Using this approach on an environmental sample from the Patagonian Shelf, we produced three distinct libraries. One of the virus libraries was dominated (79.65% of sequences with known viral homology) by giant viruses from the Mimiviridae and Phycodnaviridae families, while the two other viromes were dominated by smaller phycodnaviruses, cyanophages and other bacteriophages. The estimated genotypic richness and diversity in our sorted viromes, with 52-163 estimated genotypes, was much lower than in previous virome reports. Fragment recruitment of metagenome reads to selected reference viral genomes yields high genome coverage, suggesting little amplification and sequencing bias against some genomic regions. These results underscore the value of our approach as an effective way to target and investigate specific virus groups. In particular, it will help reveal the diversity and abundance of giant viruses in marine ecosystems.
宏基因组学为研究病毒多样性和功能开辟了新的窗口。病毒组学研究通常需要大量的样本量,并通过 0.2μm 孔径的过滤器进行过滤,因此会排除或采样不足长尾和非常大的病毒。我们优化了一种靶向病毒组学方法,该方法采用荧光激活分选和全基因组扩增,从 1 毫升水样中的离散病毒群中产生富含 dsDNA 的文库。使用这种方法对巴塔哥尼亚大陆架的环境样本进行分析,我们产生了三个不同的文库。其中一个病毒文库主要由 Mimiviridae 和 Phycodnaviridae 家族的巨型病毒组成(具有已知病毒同源性的序列占 79.65%),而另外两个病毒组则主要由较小的藻病毒、噬藻体和其他噬菌体组成。我们分选的病毒组中的估计基因型丰富度和多样性为 52-163 个估计基因型,远低于以前的病毒组报告。从选定的参考病毒基因组中招募宏基因组序列的片段,可获得高基因组覆盖率,表明某些基因组区域的扩增和测序偏差很小。这些结果强调了我们的方法作为一种有效靶向和研究特定病毒群的方法的价值。特别是,它将有助于揭示海洋生态系统中巨型病毒的多样性和丰度。