Capturing native RNA 3' ends by poly-inosine tailing and direct RNA sequencing on a nanopore

The standard protocol for direct RNA sequencing using Oxford Nanopore Technology's (ONT) system is designed to capture RNAs ending in a poly(A) tail. Sequencing of non-poly(A) RNAs is possible through the use of custom adaptors or by adding poly(A) to RNA 3' ends before preparing the sample for sequencing. There are a number of natural post-transcriptional processes that add A residues to RNA, and thus the current sequencing protocol destroys important biological information in the sample. addition of A residues to the end of an RNA obscures alters the 3' end of the natural RNA. To preserve and capture native 3' end nucleotide sequence, we developed a poly-inosine (polyI) tailing method that preserves the natural 3' end structure while allowing the addition of sequencing adaptors. We show that most polyA and non-polyA RNAs acquire about 38 inosine residues, and that the inosine homopolymer produces a distinctive current trace in the pore that allows it to be distinguished from a natural polyA tail. We have applied this method to RNA samples from yeast and human cells, and compare the effectiveness and reproducibility of polyI-tailing to the standard method. Poly-I tailing allows 3' ends of RNA to be accurately characterized, and can enable discovery of new non-polyadenylated noncoding RNAs.