Enriching pathogen transcripts from infected samples: A capture-based approach to enhanced host-pathogen RNA sequencing
Enriching pathogen transcripts from infected samples: A capture-based approach to enhanced host-pathogen RNA sequencing
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DOI：
10.1016/j.ab.2013.03.008
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发表时间：
2013-07-01
影响因子：
2.9
通讯作者：
VanderNoot, Victoria A.
中科院分区：
文献类型：
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作者：
Bent, Zachary W.;Tran-Gyamfi, Mary B.;VanderNoot, Victoria A.
为了充分了解病原体与其宿主的相互作用，有必要在整个感染过程中分析宿主和病原体的RNA转录本。虽然这可以相对容易地在宿主侧完成，但病原体转录物的分析由于从感染的样品中分离的大量宿主RNA而变得复杂。即使使用第二代测序提供的读取深度，也极难获得足够的病原体读取以进行有效的基因水平分析。在这项研究中，我们描述了一种新的捕获为基础的技术和设备，大大丰富了病原体转录从感染的样本。原则上，这种通用方法可以富集任何感染样品中的任何病原体。为了测试该技术的有效性，我们使用裂谷热病毒和土拉热弗朗西斯菌进行了时程组织培养感染。在每个时间点，进行RNA测序（RNA-Seq），并将处理的样品的结果与未处理的对照进行比较。在所有情况下，病原体转录物的捕获导致病原体读段的数量级以上的富集，大大增加了命中的基因数量、这些基因的覆盖范围以及每个转录物测序的深度。(C)2013 Elsevier Inc. All rights reserved.
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. (C) 2013 Elsevier Inc. All rights reserved.