Identification of high-confidence human poly(A) RNA isoform scaffolds using nanopore sequencing

RNA. 2022 Feb;28(2):162-176. doi: 10.1261/rna.078703.121. Epub 2021 Nov 2.


Nanopore sequencing devices read individual RNA strands directly. This facilitates identification of exon linkages and nucleotide modifications; however, using conventional direct RNA nanopore sequencing, the 5' and 3' ends of poly(A) RNA cannot be identified unambiguously. This is due in part to RNA degradation in vivo and in vitro that can obscure transcription start and end sites. In this study, we aimed to identify individual full-length human RNA isoforms among ∼4 million nanopore poly(A)-selected RNA reads. First, to identify RNA strands bearing 5' m7G caps, we exchanged the biological cap for a modified cap attached to a 45-nt oligomer. This oligomer adaptation method improved 5' end sequencing and ensured correct identification of the 5' m7G capped ends. Second, among these 5'-capped nanopore reads, we screened for features consistent with a 3' polyadenylation site. Combining these two steps, we identified 294,107 individual high-confidence full-length RNA scaffolds from human GM12878 cells, most of which (257,721) aligned to protein-coding genes. Of these, 4876 scaffolds indicated unannotated isoforms that were often internal to longer, previously identified RNA isoforms. Orthogonal data for m7G caps and open chromatin, such as CAGE and DNase-HS seq, confirmed the validity of these high-confidence RNA scaffolds.

Keywords: RNA; RNA isoforms; direct RNA; full-length; nanopore; single-molecule; transcription start site; transcriptomics.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Cell Line, Tumor
  • Humans
  • Nanopore Sequencing / methods
  • RNA 3' Polyadenylation Signals
  • RNA Isoforms / chemistry*
  • RNA Isoforms / genetics
  • RNA, Messenger / chemistry*
  • RNA, Messenger / genetics
  • Transcriptome


  • RNA Isoforms
  • RNA, Messenger