Modification mapping by nanopore sequencing.
Modification mapping by nanopore sequencing.
复制标题
DOI：
10.3389/fgene.2022.1037134
复制
发表时间：
2022
影响因子：
3.7
通讯作者：
中科院分区：
文献类型：
--
作者：
在过去的20年里，下一代测序(NGS)为生物学家提供了一个前所未有的视角来了解生物过程及其调控，推动了基于短读DNA和RNA测序的高通量方法的发展。在核酸修饰方面，NGS与免疫沉淀、化学处理、酶处理和/或具有偶然活性的逆转录酶的使用相结合，以丰富和鉴定RNA和DNA的共价修饰。然而，大多数核酸修饰缺乏商品化的单抗，而依赖化学或酶处理来操纵修饰特征的作图技术增加了文库制备的技术复杂性。此外，这种方法倾向于针对单一类别的RNA或DNA修饰，并且只产生修饰状态的间接读数。第三代测序技术，如太平洋生物科学公司和牛津纳米孔技术公司的商业化“长读”平台，是高通量检测核酸修饰的有吸引力的替代方案。前者可以通过逆转录反应动力学的变化间接检测修饰的核苷酸，而纳米孔测序原则上可以直接检测任何在核酸通过嵌入带电薄膜的纳米孔传感器时产生信号失真的核酸修饰。到目前为止，已有十几种内源性DNA和RNA修饰被纳米孔测序以及一些用于代谢标记、结构探测和其他新兴应用的合成核酸修饰所询问。这篇综述旨在向读者介绍纳米孔测序及其在直接检测未扩增DNA或RNA样品中的核酸修饰方面的基本原理，并概述当前通过纳米孔测序检测和定量核酸修饰的方法。随着这项技术的成熟，我们预计测序化学和分析方法的进步将导致这些表观遗传标记的识别和量化方面的快速改进。
Next generation sequencing (NGS) has provided biologists with an unprecedented view into biological processes and their regulation over the past 2 decades, fueling a wave of development of high throughput methods based on short read DNA and RNA sequencing. For nucleic acid modifications, NGS has been coupled with immunoprecipitation, chemical treatment, enzymatic treatment, and/or the use of reverse transcriptase enzymes with fortuitous activities to enrich for and to identify covalent modifications of RNA and DNA. However, the majority of nucleic acid modifications lack commercial monoclonal antibodies, and mapping techniques that rely on chemical or enzymatic treatments to manipulate modification signatures add additional technical complexities to library preparation. Moreover, such approaches tend to be specific to a single class of RNA or DNA modification, and generate only indirect readouts of modification status. Third generation sequencing technologies such as the commercially available “long read” platforms from Pacific Biosciences and Oxford Nanopore Technologies are an attractive alternative for high throughput detection of nucleic acid modifications. While the former can indirectly sense modified nucleotides through changes in the kinetics of reverse transcription reactions, nanopore sequencing can in principle directly detect any nucleic acid modification that produces a signal distortion as the nucleic acid passes through a nanopore sensor embedded within a charged membrane. To date, more than a dozen endogenous DNA and RNA modifications have been interrogated by nanopore sequencing, as well as a number of synthetic nucleic acid modifications used in metabolic labeling, structure probing, and other emerging applications. This review is intended to introduce the reader to nanopore sequencing and key principles underlying its use in direct detection of nucleic acid modifications in unamplified DNA or RNA samples, and outline current approaches for detecting and quantifying nucleic acid modifications by nanopore sequencing. As this technology matures, we anticipate advances in both sequencing chemistry and analysis methods will lead to rapid improvements in the identification and quantification of these epigenetic marks.
影响因子：
17.1
作者：
Clamer M;Höfler L;Mikhailova E;Viero G;Bayley H
通讯作者：
Bayley H