Nanopore-based direct sequencing of RNA transcripts avoids the limitations imposed by reverse<u> </u>transcription in cDNA sequencing and provides the potential for ultra-long reads and identification of nucleotide modifications. The current methodology, however, does not allow identification of full-length reads of any RNA transcript. Upon reaching the 5’ end of the RNA, the motor protein releases the RNA, which causes the last approximately 20 nucleotides to slip quickly through the pore with their sequence information lost. Obtaining the complete RNA sequence through the 5’ end is critical in order to identify transcription start sites (TSSs) and their linkage to mRNA splice variants. We have developed methods to capture the 5’ ends of primary mRNA transcripts and use them as handles for enrichment, sequencing, and TSS determination. Here we report Nanopore ReCappable-Seq, a method that enables modification and extension of the 5’ end capped RNA molecules for nanopore direct full-length RNA sequencing. We employed enzymatic decapping and recapping with a GTP analog that can be coupled to an adaptor using click chemistry to yeast and human poly(A) mRNAs. MinION sequencing of these samples revealed improved TSS coverage and identification of alternative TSS. This methodology provides complete full-length sequencing of the RNA ends and thus allows for the correct identification of isoforms.

Identifying full-length transcripts in eukaryotic mRNA using direct RNA nanopore sequencing

Macromolecular Chemistry: The Second Century

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ACS Spring 2020 National Meeting & Expo

The official archive and repository for all ACS Meetings & Expositions abstracts and select posters or presentations.