RT Journal Article
SR Electronic
T1 AQ-seq: Accurate quantification of microRNAs and their variants
JF bioRxiv
FD Cold Spring Harbor Laboratory
SP 339606
DO 10.1101/339606
A1 Haedong Kim
A1 Jimi Kim
A1 Kijun Kim
A1 Hyeshik Chang
A1 Kwontae You
A1 V. Narry Kim
YR 2018
UL http://biorxiv.org/content/early/2018/06/05/339606.abstract
AB MicroRNAs (miRNAs) modulate diverse biological and pathological processes via post-transcriptional 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 sRNA-seq 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).