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.
Researchers at New England Biolabs have 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. They 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, the researchers 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.
Schematic of randomized splint ligation library preparation
First the preadenylated 3′ adapter is ligated on using randomized splint ligation. Following adapter ligation, the excess adapter is depleted using 5′ deadenylase and lambda exonuclease, and the degenerate portion of the adapter is cleaved off by excising the deoxyuracil using USER. Next the 5′ adapter is ligated on using randomized splint ligation and cDNA is synthesized using the remaining portion of the 3′ adapter splint strand as a primer for the reverse transcription. Finally, library molecules containing both adapters are enriched and extended using PCR.