True end-to-end sequencing of single native RNA molecules with TERA-Seq

Direct sequencing of single, native RNA molecules and their modifications through nanopores has a strong potential to transform research in all aspects of RNA biology and for clinical diagnostics. The existing platform from Oxford Nanopore Technologies is unable to reliably capture the 5' ends of molecules and is limited to sequencing of primarily polyadenylated molecules. Here, we develop True End-to-end RNA Sequencing (TERA-Seq) by ligating adapters to the 5' and 3' ends of single RNA molecules, which are then sequenced along with the native transcript. We find that capped full-length mRNAs in human show marked variation of poly(A) tail lengths at the single-molecule resolution. We report prevalent capping downstream of canonical transcriptional initiation sites in otherwise fully spliced and polyadenylated molecules. We reveal RNA processing and decay at the single molecules level and find that mRNAs decay cotranslationally, often while retaining poly(A) tails. We envision that TERA-Seq will prove useful in many applications where end-to-end sequencing of single, native RNA molecules and their modifications is desirable.