SHAPE-Seq 2.0: systematic optimization and extension of high-throughput chemical probing of RNA secondary structure with next generation sequencing.
SHAPE-Seq 2.0: systematic optimization and extension of high-throughput chemical probing of RNA secondary structure with next generation sequencing.
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DOI：
10.1093/nar/gku909
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发表时间：
2014-12-01
影响因子：
14.9
通讯作者：
Lucks JB
中科院分区：
文献类型：
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作者：
Loughrey D;Watters KE;Settle AH;Lucks JB
RNA 结构是其功能的主要决定因素，将化学探测与下一代测序相结合的方法在 RNA 结构表征的通量和规模方面取得了突破。然而，很少有人研究文库制备和测序对编码 RNA 结构信息的化学探针反应性的影响。在这里，我们首次对通过引物延伸测序 (SHAPE-Seq) 分析的选择性 2'-羟基酰化的影响进行了分析和优化。我们首先优化 SHAPE-Seq，并证明它可以在广泛的 RNA 结构背景下提供高度可重复的反应性数据，没有明显的偏差。作为优化的一部分，我们推出了 SHAPE-Seq v2.0，这是一种“通用”方法，可以获取 RNA 每个核苷酸的反应性信息，而无需在 RNA 中使用或引入特定的逆转录酶引发位点。我们证明 SHAPE-Seq v2.0 具有高度可重复性，其反应性数据可用作 RNA 折叠算法的约束，以预测与使用其他 SHAPE 方法的数据生成的结构相当的结构。我们预计 SHAPE-Seq v2.0 将广泛适用于理解生命中一些最基本过程的核心 RNA 序列-结构关系。
RNA structure is a primary determinant of its function, and methods that merge chemical probing with next generation sequencing have created breakthroughs in the throughput and scale of RNA structure characterization. However, little work has been done to examine the effects of library preparation and sequencing on the measured chemical probe reactivities that encode RNA structural information. Here, we present the first analysis and optimization of these effects for selective 2′-hydroxyl acylation analyzed by primer extension sequencing (SHAPE-Seq). We first optimize SHAPE-Seq, and show that it provides highly reproducible reactivity data over a wide range of RNA structural contexts with no apparent biases. As part of this optimization, we present SHAPE-Seq v2.0, a ‘universal’ method that can obtain reactivity information for every nucleotide of an RNA without having to use or introduce a specific reverse transcriptase priming site within the RNA. We show that SHAPE-Seq v2.0 is highly reproducible, with reactivity data that can be used as constraints in RNA folding algorithms to predict structures on par with those generated using data from other SHAPE methods. We anticipate SHAPE-Seq v2.0 to be broadly applicable to understanding the RNA sequence–structure relationship at the heart of some of life's most fundamental processes.
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DOI：
10.1007/978-1-62703-113-4_7
发表时间：
2012-01-01
期刊：
Methods in molecular biology (Clifton, N.J.)
影响因子：
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作者：
Avis, Johanna M;Conn, Graeme L;Walker, Scott C
通讯作者：
Walker, Scott C
影响因子：
4.5
作者：
Karabiber, Fethullah;McGinnis, Jennifer L.;Weeks, Kevin M.
通讯作者：
Weeks, Kevin M.
DOI：
10.1073/pnas.1219988110
发表时间：
2013-04-02
影响因子：
11.1
作者：
Hajdin, Christine E.;Bellaousov, Stanislav;Weeks, Kevin M.
通讯作者：
Weeks, Kevin M.
影响因子：
2.9
作者：
Tyrrell J;McGinnis JL;Weeks KM;Pielak GJ
通讯作者：
Pielak GJ
影响因子：
64.8
作者：
通讯作者：
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