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首页> 外文期刊>Journal of Molecular Biology >ClickSeq: Fragmentation-Free Next-Generation Sequencing via Click Ligation of Adaptors to Stochastically Terminated 3 '-Azido cDNAs
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ClickSeq: Fragmentation-Free Next-Generation Sequencing via Click Ligation of Adaptors to Stochastically Terminated 3 '-Azido cDNAs

机译：ClickSeq：通过衔接子与随机终止的3'-叠氮基cDNA的点击连接，实现无片段化的下一代测序

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摘要

We present a simple method called "ClickSeq" for NGS (next-generation sequencing) library synthesis that uses click chemistry rather than enzymatic reactions for the ligation of Illumina sequencing adaptors. In ClickSeq, randomly primed reverse transcription reactions are supplemented with azido-2',3'-dideoxynucleotides that randomly terminate DNA synthesis and release 3'-azido-blocked cDNA fragments in a process akin to dideoxy-Sanger sequencing. Purified fragments are "click ligated" via copper-catalyzed alkyne-azide cycloaddition to DNA oligos modified with a 5'-alkyne group. This generates ssDNA molecules containing an unnatural triazole-linked DNA backbone that is sufficiently biocompatible for PCR amplification to generate a cDNA library for RNAseq. Here, we analyze viral RNAs and mRNA to demonstrate that ClickSeq produces unbiased NGS libraries with low error rates comparable to standard methods. Importantly, ClickSeq is robust against common artifacts of NGS such as chimera formation and artifactual recombination with fewer than 3 aberrant events detected per million reads. (C) 2015 Elsevier Ltd. All rights reserved.
机译：我们为NGS（下一代测序）库合成提供了一种称为“ ClickSeq”的简单方法，该方法使用点击化学而非酶促反应来连接Illumina测序适配器。在ClickSeq中，随机引发的逆转录反应辅以azido-2'，3'-dideoxynucleotides，后者可随机终止DNA合成并释放3'-azido-blocked cDNA片段，类似于双脱氧-Sanger测序。纯化的片段通过铜催化的炔-叠氮化物环加成“点击连接”到经5'-炔基修饰的DNA寡聚体上。这产生了包含非天然三唑连接的DNA主链的ssDNA分子，该主链具有足够的生物相容性以进行PCR扩增，从而生成RNAseq的cDNA文库。在这里，我们分析了病毒RNA和mRNA，以证明ClickSeq可以产生无偏的NGS文库，其错误率可与标准方法相比。重要的是，ClickSeq可以抵抗NGS的常见伪影，例如嵌合体形成和伪影重组，每百万次读取检测到少于3个异常事件。 （C）2015 Elsevier Ltd.保留所有权利。

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