Sandia National Laboratories, Livermore, CA 94551, USA.
Anal Biochem. 2013 Jul 1;438(1):90-6. doi: 10.1016/j.ab.2013.03.008. Epub 2013 Mar 25.
To fully understand the interactions of a pathogen with its host, it is necessary to analyze the RNA transcripts of both the host and pathogen throughout the course of an infection. Although this can be accomplished relatively easily on the host side, the analysis of pathogen transcripts is complicated by the overwhelming amount of host RNA isolated from an infected sample. Even with the read depth provided by second-generation sequencing, it is extremely difficult to get enough pathogen reads for an effective gene-level analysis. In this study, we describe a novel capture-based technique and device that considerably enriches for pathogen transcripts from infected samples. This versatile method can, in principle, enrich for any pathogen in any infected sample. To test the technique's efficacy, we performed time course tissue culture infections using Rift Valley fever virus and Francisella tularensis. At each time point, RNA sequencing (RNA-Seq) was performed and the results of the treated samples were compared with untreated controls. The capture of pathogen transcripts, in all cases, led to more than an order of magnitude enrichment of pathogen reads, greatly increasing the number of genes hit, the coverage of those genes, and the depth at which each transcript was sequenced.
为了充分了解病原体与其宿主之间的相互作用，有必要在感染过程中分析宿主和病原体的 RNA 转录本。尽管在宿主方面相对容易完成此操作，但由于从感染样本中分离出的宿主 RNA 数量巨大，因此对病原体转录本的分析变得复杂。即使使用第二代测序提供的读取深度，也很难获得足够的病原体读取量来进行有效的基因水平分析。在这项研究中，我们描述了一种新颖的基于捕获的技术和设备，可从感染样本中大量富集病原体转录本。这种多功能方法原则上可以从任何感染样本中富集任何病原体。为了测试该技术的功效，我们使用裂谷热病毒和土拉弗朗西斯菌进行了时间过程组织培养感染。在每个时间点，都进行了 RNA 测序（RNA-Seq），并将处理过的样本的结果与未经处理的对照进行了比较。在所有情况下，病原体转录本的捕获导致病原体读取数增加了一个数量级以上，大大增加了命中的基因数量、这些基因的覆盖度以及每个转录本的测序深度。
