AQ-seq: Accurate quantification of microRNAs and their variants

Abstract

MicroRNAs (miRNAs) modulate diverse biological and pathological processes via posttranscriptional gene silencing. High-throughput small RNA sequencing (sRNA-seq) has been widely adopted to investigate the functions and regulatory mechanisms of miRNAs. However, accurate quantification has been limited owing to the severe ligation bias in conventional sRNAseq methods. Here we present a high-throughput protocol, termed AQ-seq (accurate quantification by sequencing), that utilizes adapters with terminal degenerate sequences and a high concentration of polyethylene glycol (PEG), which removes the ligation bias during library preparation. By accurately measuring miRNAs and their variants (known as isomiRs), we identify alternatively processed miRNAs and correct the 5′ end usage and strand preference that have been misannotated. We also uncover highly modified miRNAs that are uridylated and adenylated.Taken together, AQ-seq reveals the complexity of the miRNA isoform landscape, allowing us to refine miRNA annotation and to advance our understanding of miRNA regulation. Furthermore, AQ-seq can be adopted to improve other ligation-based sequencing methods including crosslinking-immunoprecipitation-sequencing (CLIP-seq) and ribosome profiling (Ribo-seq).

Unification of miRNA and isomiR research: the mirGFF3 format and the mirtop API

“…IsomiRs are sequence variants from annotated miRNAs [19][20][21]. IsomiRs were first described in detail by Morin et al [22] in human stem cell lines using next generation sequencing technologies.…”

Molecular Basis for the Single-Nucleotide Precision of Primary microRNA Processing

“…Small RNA sequencing libraries were prepared by following our recently developed protocol (Kim et al, 2018a) to reduce ligation bias. Briefly, total RNA was purified using TRIzol (Thermo Fisher), and 10 mg of total RNA was mixed with 30 spike-in RNAs; then, 18-30-nt fragments were purified through a 15% urea-polyacrylamide gel.…”