Quantitative and Multiplexing Analysis of MicroRNAs by Direct Full-Length Sequencing in Nanopores

MicroRNAs (miRNAs) play important regulatory roles in biology. Direct sequencing of miRNAs in full-length can reveal comprehensive information on their sequences, abundance, and modifications, which, however, has yet to be achieved due to their extremely short length (∼22 nt). Herein, we developed Direct-miR-seq, a nanopore-based direct RNA sequencing (DRS) method that elongates miRNAs at both the 5' and 3' ends by ligating with custom nucleic acid adaptors to ensure full-length sequencing of miRNAs with high yield and accuracy. Compared to standard DRS, Direct-miR-seq enabled sequencing of the whole sequence of miRNAs, achieved a 26-fold sequencing yield, and exhibited reduced bias across miRNA species along with low sequencing error rates. We applied Direct-miR-seq to native RNA populations from cells and human serum to demonstrate its capability to selectively capture miRNAs of known sequences in complex RNA environments for revealing quantitative information in abundance and m6A modification at single-molecule and single-base resolution of ∼100 miRNA species in a single sequencing event. We envision that Direct-miR-seq may be translated toward a variety of biological and medical applications by sequencing miRNAs and other small RNAs.