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

Nanopore sequencing devices read individual RNA strands directly. This facilitates identification of exon linkages and nucleotide modifications; however, using conventional methods the 5' and 3' ends of poly(A) RNA cannot be identified unambiguously. This is due in part to the architecture of the nanopore/enzyme-motor complex, and 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 isoform scaffolds 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-nucleotide 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 ionic current signatures consistent with a 3' polyadenylation site.

Combining these two steps, we identified 294,107 individual high-confidence full-length RNA scaffolds, most of which (257,721) aligned to protein-coding genes. Of these, 4,876 scaffolds indicated unannotated isoforms that were often internal to longer, previously identified RNA isoforms. Orthogonal data confirmed the validity of these high-confidence RNA scaffolds.