The utility of shallow RNA-Seq for documenting differential gene expression in genes with high and low levels of expression.
The utility of shallow RNA-Seq for documenting differential gene expression in genes with high and low levels of expression.
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DOI：
10.1371/journal.pone.0084160
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发表时间：
2013
期刊：
影响因子：
3.7
通讯作者：
Johnson BR
中科院分区：
文献类型：
--
作者：
Atallah J;Plachetzki DC;Jasper WC;Johnson BR
使用RNA-Seq记录差异基因表达所需的测序深度在模型系统之外很少被探索。特别是，分析大规模差异转录因子表达模式所需的深度尚不清楚。本研究的目的是探索浅层（相对较低的读取深度）RNA-Seq的有效性。我们关注蜜蜂的两个组织：刺腺和消化道。刺腺是一种实验上很好理解的组织，我们用它来衡量这种方法的实用性。我们使用消化道来测试刺腺获得的结果，并在组织类型之间进行RNA-Seq。使用实验验证的基因列表赋予组织特异性功能在刺腺，我们表明，相对较少的阅读深度是必要的，以确定他们。我们认为这一结果应该广泛适用，因为对组织特异性功能重要的基因通常具有强大的表达模式，并且因为我们在消化道分析中获得了类似的结果。此外，我们证明了转录因子的差异表达，与其他基因相比转录水平较低，但通常可以使用浅RNA-Seq来确定。总的来说，我们在我们的组织中发现了150多个差异表达的转录因子，读取深度仅为1200万。这项工作显示了低深度测序在识别对组织特异性功能重要的基因方面的效用。它还验证了人们通常持有的观点，即转录因子的表达水平较低，同时表明，尽管如此，它们通常适用于浅RNA-Seq。我们的发现应该对在许多不同的生物系统中使用RNA-Seq的研究人员有益。
The sequencing depth necessary for documenting differential gene expression using RNA-Seq has been little explored outside of model systems. In particular, the depth required to analyze large-scale patterns of differential transcription factor expression is not known. The goal of the present study is to explore the effectiveness of shallow (relatively low read depth) RNA-Seq. We focus on two tissues in the honey bee: the sting gland and the digestive tract. The sting gland is an experimentally well-understood tissue that we use to benchmark the utility of this approach. We use the digestive tract to test the results obtained with the sting gland, and to conduct RNA-Seq between tissue types. Using a list of experimentally verified genes conferring tissue-specific functions in the sting gland, we show that relatively little read depth is necessary to identify them. We argue that this result should be broadly applicable, since genes important for tissue-specific functions often have robust expression patterns, and because we obtained similar results in our analysis of the digestive tract. Furthermore, we demonstrate that the differential expression of transcription factors, which are transcribed at low levels compared to other genes, can nevertheless often be determined using shallow RNA-Seq. Overall, we find over 150 differentially expressed transcription factors in our tissues at a read depth of only 12 million. This work shows the utility of low-depth sequencing for identifying genes important for tissue-specific functions. It also verifies the often-held belief that transcription factors show low levels of expression, while demonstrating that, in spite of this, they are frequently amenable to shallow RNA-Seq. Our findings should be of benefit to researchers using RNA-Seq in many different biological systems.
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