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首页> 美国卫生研究院文献>Journal of Biomolecular Techniques : JBT >ScriptSeq RNA-Seq Library Preparation Method: A Simplified Work-Flow for Directional NGS RNASeq Library Preparation with Whole-Transcript Representation
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ScriptSeq RNA-Seq Library Preparation Method: A Simplified Work-Flow for Directional NGS RNASeq Library Preparation with Whole-Transcript Representation

机译：ScriptSeq RNA-Seq文库制备方法：具有完整转录本表示的定向NGS RNASeq文库制备的简化工作流程

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RNA sequencing is an emerging revolutionary tool for whole transcriptome analysis that provides information about the structure of transcripts and their expression levels. Current methods for making sequencer-specific di-tagged DNA fragment libraries for RNA-Seq typically comprise preparing rRNA-depleted RNA and either (i) RNA fragmentation, 5' and 3' adaptor-ligation, size selection, cDNA synthesis, and multiple clean-up steps; or (ii) cDNA synthesis followed by cDNA fragmentation, end-polishing, 5' and 3' adaptor-ligation, size selection and multiple clean-up steps. These methods are generally long and require significant hands-on time. We describe a novel protocol that utilizes a unique Terminal-Tagging technology that simplifies the preparation of directional RNA-Seq libraries from rRNA-depleted or poly(A)-enriched RNA in about 3 hours, without the need for adaptor ligation, cDNA nebulization or gel purification. The di-tagged cDNA fragments that are generated from this simple, single tube protocol are compatible with different NGS sequencing platforms. Apart from its simplicity, another major strength of this RNA-seq protocol is its ability to determine the polarity of the RNA transcripts, which is critical for the annotation of novel genes. Sequencing results obtained from libraries prepared by using RiboZero™ rRNA-depletion method and ScriptSeq™ mRNA-Seq Library Preparation Method show excellent directionality with less than 2 % of the sequence reads that map to rRNA sequences (28S, 18S, 5.8S and 5S). This reduction in rRNA sequence reads improves sequence depth and coverage, and increases the percentage of uniquely mapped reads. Further, there is a high correlation (R2=0.9235) between differentially expressed transcripts found in the ScriptSeq™ RNA-Seq libraries and the MAQC QPCR panel of genes.
机译：RNA测序是用于整体转录组分析的新兴革命性工具，可提供有关转录本结构及其表达水平的信息。制备用于RNA-Seq的序列特异性双标签DNA片段文库的当前方法通常包括制备耗尽rRNA的RNA和（i）RNA片段化，5'和3'衔接子连接，大小选择，cDNA合成以及多次纯化-上一步；或（ii）cDNA合成，然后进行cDNA片段化，末端抛光，5'和3'衔接子连接，大小选择和多个清理步骤。这些方法通常很长，并且需要大量的动手时间。我们描述了一种新颖的协议，该协议利用了独特的终端标记技术，可简化大约3小时内从rRNA耗尽或富含poly（A）的RNA中制备定向RNA-Seq库的过程，而无需适配器连接，cDNA雾化或凝胶纯化。通过这种简单的单管方案生成的双标签cDNA片段与不同的NGS测序平台兼容。除了简单之外，这种RNA-seq协议的另一个主要优势是它能够确定RNA转录本的极性，这对于注释新基因至关重要。从使用RiboZero™rRNA耗竭方法和ScriptSeq™mRNA-Seq库制备方法制备的文库中获得的测序结果显示出优异的方向性，少于映射到rRNA序列（28S，18S，5.8S和5S）的2％的序列读数。 rRNA序列读段的减少可改善序列深度和覆盖范围，并增加唯一映射读段的百分比。此外，在ScriptSeq™RNA-Seq文库中发现的差异表达转录物与MAQC QPCR基因组之间具有高度相关性（R2 = 0.9235）。

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