Australian Institute of Marine Science Townsville, QLD, Australia.
Department of Microbiology and Immunology, University of British Columbia Vancouver, BC, Canada ; Department of Earth, Ocean and Atmospheric Sciences, University of British Columbia Vancouver, BC, Canada ; Department of Botany, University of British Columbia Vancouver, BC, Canada ; Canadian Institute for Advanced Research, University of British Columbia Vancouver, BC, Canada.
Front Microbiol. 2014 May 7;5:206. doi: 10.3389/fmicb.2014.00206. eCollection 2014.
Reef-building corals comprise multipartite symbioses where the cnidarian animal is host to an array of eukaryotic and prokaryotic organisms, and the viruses that infect them. These viruses are critical elements of the coral holobiont, serving not only as agents of mortality, but also as potential vectors for lateral gene flow, and as elements encoding a variety of auxiliary metabolic functions. Consequently, understanding the functioning and health of the coral holobiont requires detailed knowledge of the associated viral assemblage and its function. Currently, the most tractable way of uncovering viral diversity and function is through metagenomic approaches, which is inherently difficult in corals because of the complex holobiont community, an extracellular mucus layer that all corals secrete, and the variety of sizes and structures of nucleic acids found in viruses. Here we present the first protocol for isolating, purifying and amplifying viral nucleic acids from corals based on mechanical disruption of cells. This method produces at least 50% higher yields of viral nucleic acids, has very low levels of cellular sequence contamination and captures wider viral diversity than previously used chemical-based extraction methods. We demonstrate that our mechanical-based method profiles a greater diversity of DNA and RNA genomes, including virus groups such as Retro-transcribing and ssRNA viruses, which are absent from metagenomes generated via chemical-based methods. In addition, we briefly present (and make publically available) the first paired DNA and RNA viral metagenomes from the coral Acropora tenuis.
造礁珊瑚形成多组分共生关系，其中刺胞动物是一系列真核和原核生物以及感染它们的病毒的宿主。这些病毒是珊瑚整体共生体的关键组成部分，不仅是致死因子，而且还可能作为横向基因流动的潜在载体，并编码各种辅助代谢功能。因此，要了解珊瑚整体共生体的功能和健康状况，就需要详细了解相关的病毒组合及其功能。目前，揭示病毒多样性和功能的最可行方法是通过宏基因组学方法，由于复杂的整体共生群落、所有珊瑚分泌的细胞外黏液层以及病毒中发现的各种大小和结构的核酸，这种方法在珊瑚中固有地具有挑战性。在这里，我们提出了一种基于细胞机械破坏从珊瑚中分离、纯化和扩增病毒核酸的新方法。该方法产生的病毒核酸产量至少高出 50%，细胞序列污染水平非常低，并且比以前使用的基于化学的提取方法捕获了更广泛的病毒多样性。我们证明，我们的基于机械的方法可以分析出更多种类的 DNA 和 RNA 基因组，包括逆转录病毒和单链 RNA 病毒等病毒群，这些病毒群在基于化学的方法生成的宏基因组中不存在。此外，我们简要介绍了（并公开提供）来自软珊瑚 Acropora tenuis 的第一个配对的 DNA 和 RNA 病毒宏基因组。
