A new approach to determining whole viral genomic sequences including termini using a single deep sequencing run
A new approach to determining whole viral genomic sequences including termini using a single deep sequencing run
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DOI：
10.1016/j.jviromet.2014.07.023
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发表时间：
2014-11-01
影响因子：
3.1
通讯作者：
Griffiths, Anthony
中科院分区：
文献类型：
--
作者：
Alfson, Kendra J.;Beadles, Michael W.;Griffiths, Anthony
新一代测序现已广泛应用于病毒学领域，包括病毒发现、准种研究、病毒进化、宏基因组学和抗病毒药物耐药性分析。然而，目前用于病毒基因组深度测序的样品制备方法存在局限性，特别是在从头测序过程中。例如，目前的方法无法高效地捕获病毒基因组的末端序列；代表3 ‘和5 ’两端的数据通常是不足的。快速扩增cDNA末端等方法解决了这个问题，但这些方法可能耗时，可能需要一些病毒序列的先验知识，并且需要多个独立的程序。目前的研究概述了一种样品制备技术，克服了这些缺点。该方法依赖于二价阳离子的随机片段化和随后直接连接到RNA的适配器，而不是cDNA，以最大限度地提高末端读取的质量和数量。这项技术在埃博拉病毒和丙型肝炎病毒两种不同RNA病毒的RNA样本上进行了测试。该方法允许快速制备样品进行深度测序，同时消除了序列特异性引物的使用，并捕获了整个基因组序列，包括5 ‘和3 ’端。这可以提高终端未知的病毒发现项目的效率。(C) 2014 Elsevier B.V.版权所有
Next-generation sequencing is now commonly used for a variety of applications in virology including virus discovery, investigation of quasispecies, viral evolution, metagenomics, and analyses of antiviral resistance. However, there are limitations with the current sample preparation methods used for deep sequencing of viral genomes, especially during de novo sequencing. For example, current methods are unable to capture the terminal sequences of viral genomes in an efficient and effective manner; data representing the 3 ' and 5 ' ends are typically insufficient. Methods such as Rapid Amplification of cDNA Ends address this issue but these methods can be time consuming, may require some prior knowledge of the viral sequence, and require multiple independent procedures. The current study outlines a sample preparation technique that overcomes some of these shortcomings. The method relied on random fragmentation with divalent cations and subsequent adapter ligation directly to RNA, rather than cDNA, to maximize the quality and quantity of terminal reads. The technique was tested on RNA samples from two different RNA viruses, Ebola virus and hepatitis C virus. This method permits rapid preparation of samples for deep sequencing while eliminating the use of sequence specific primers and captures the entire genome sequence, including the 5 ' and 3 ' ends. This could improve the efficiency of virus discovery projects where the terminal ends are unknown. (C) 2014 Elsevier B.V. All rights reserved.