New England Biolabs, Inc., 240 County Road, Ipswich, MA 01938, USA.
Nucleic Acids Res. 2020 Aug 20;48(14):e80. doi: 10.1093/nar/gkaa480.
Small RNAs are important regulators of gene expression and are involved in human development and disease. Next generation sequencing (NGS) allows for scalable, genome-wide studies of small RNA; however, current methods are challenged by low sensitivity and high bias, limiting their ability to capture an accurate representation of the cellular small RNA population. Several studies have shown that this bias primarily arises during the ligation of single-strand adapters during library preparation, and that this ligation bias is magnified by 2'-O-methyl modifications (2'OMe) on the 3' terminal nucleotide. In this study, we developed a novel library preparation process using randomized splint ligation with a cleavable adapter, a design which resolves previous challenges associated with this ligation strategy. We show that a randomized splint ligation based workflow can reduce bias and increase the sensitivity of small RNA sequencing for a wide variety of small RNAs, including microRNA (miRNA) and tRNA fragments as well as 2'OMe modified RNA, including Piwi-interacting RNA and plant miRNA. Finally, we demonstrate that this workflow detects more differentially expressed miRNA between tumorous and matched normal tissues. Overall, this library preparation process allows for highly accurate small RNA sequencing and will enable studies of 2'OMe modified RNA with new levels of detail.
小 RNA 是基因表达的重要调控因子,参与人类的发育和疾病。下一代测序(NGS)允许对小 RNA 进行可扩展的全基因组研究;然而,当前的方法受到低灵敏度和高偏倚的挑战,限制了它们捕捉细胞中小 RNA 群体准确代表的能力。几项研究表明,这种偏差主要出现在文库制备过程中单链接头的连接过程中,并且这种连接偏差会被 3'末端核苷酸上的 2'-O-甲基修饰(2'OMe)放大。在这项研究中,我们开发了一种新的文库制备方法,使用带有可切割接头的随机拼接连接,该设计解决了与这种连接策略相关的先前挑战。我们表明,基于随机拼接连接的工作流程可以减少偏差并提高各种小 RNA(包括 microRNA(miRNA)和 tRNA 片段以及 2'OMe 修饰的 RNA,包括 Piwi 相互作用 RNA 和植物 miRNA)测序的灵敏度。最后,我们证明该工作流程可以检测到肿瘤和匹配正常组织之间更多差异表达的 miRNA。总体而言,该文库制备过程可实现高度准确的小 RNA 测序,并将能够以新的详细程度研究 2'OMe 修饰的 RNA。
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