一种具有更小偏差且能更好捕获 2'-O-甲基 RNA 的 Small RNA-Seq 方案。

A Small RNA-Seq Protocol with Less Bias and Improved Capture of 2'-O-Methyl RNAs.

机构信息

Institute for Integrative Biology of the Cell, UMR9198, CNRS CEA Univ Paris-Sud, Université Paris-Saclay, Gif sur Yvette Cedex, France.

出版信息

Methods Mol Biol. 2021;2298:153-167. doi: 10.1007/978-1-0716-1374-0_10.

DOI:10.1007/978-1-0716-1374-0_10

PMID:34085244

Abstract

The study of small RNAs (sRNAs) by next-generation sequencing (NGS) is challenged by bias issues during library preparation. Several types of sRNAs such as plant microRNAs (miRNAs) carry a 2'-O-methyl (2'-OMe) modification at their 3' terminal nucleotide. This modification adds another level of difficulty as it inhibits 3' adapter ligation. We previously demonstrated that modified versions of the "TruSeq (TS)" protocol have less bias and an improved detection of 2'-OMe RNAs. Here we describe in detail protocol "TS5," which showed the best overall performance. We also provide guidelines for bioinformatics analysis of the sequencing data.

摘要

下一代测序（NGS）中小 RNA（sRNA）的研究受到文库制备过程中偏倚问题的挑战。几种类型的 sRNA，如植物 microRNA（miRNA），在其 3'末端核苷酸上带有 2'-O-甲基（2'-OMe）修饰。这种修饰增加了另一个难度，因为它抑制了 3'衔接子的连接。我们之前证明，“TruSeq（TS）”协议的修饰版本具有更少的偏倚和对 2'-OMe RNA 的改进检测。在这里，我们详细描述了表现最好的协议“TS5”。我们还为测序数据的生物信息学分析提供了指导方针。