A low-bias and sensitive small RNA library preparation method using randomized splint ligation

Abstract

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.

Ligation bias is a major contributor to nonstoichiometric abundances of secondary siRNAs and impacts analyses of microRNAs

“…Most of these new protocols aim to more accurately reflect sRNA composition by focusing on reducing one identified bias, while leaving other biases unaddressed. While preparing this manuscript, a new method based on randomized splint ligation was published that involves a double-stranded adapter with a short, singlestranded degenerate extension (34). Other strategies have been developed to ensure absolute normalization of the sRNA library.…”

Transfer RNA-Derived Small RNAs: Another Layer of Gene Regulation and Novel Targets for Disease Therapeutics

“…As a result, newer forms of NGS are being developed to overcome these limitations. [53][54][55] Cozen et al 53 and Zheng et al 54 separately developed a novel library preparation method that removes methyl groups from tRNA. They used engineered E. coli AlkB to demethylate N1-methyladenosine (m1A), N3-methylcytidine (m3C), and N1methylguanosine (m1G).…”

ggVennDiagram: An Intuitive, Easy-to-Use, and Highly Customizable R Package to Generate Venn Diagram

“…Besides, Harris et al (2020) used ggVennDiagram to display that 22.5% of differentially expressed genes were shared by treated mice and human patients. Furthermore, Maguire et al (2020) used ggVennDiagram to confirm that their novel method has low bias and is more sensitive than three other methods for small RNA library preparation. In addition, ggVennDiagram is also used for analyzing the differences between several spatially varied oral metabolomics samples ( Ciurli et al, 2021 ) and for comparing single-nucleotide variants between tumor and non-tumor tissues ( Horny et al, 2021 ).…”