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, Logan Mulroney 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, Logan and his team 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.