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 unambiguous identification of full-length RNA transcripts. 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. Accurate detection of full-length transcripts is critical to identify transcription start sites (TSS) and their linkage to mRNA splice variants. We have developed a method 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 sequencing (NRCeq), a chemo-enzymatic method that enables modification and extension of the 5’ end of capped RNA molecules before nanopore sequencing. Our strategy features enzymatic decapping and recapping of native 5’ mRNA ends with a GTP analog, which is then coupled to an oligonucleotide adapter using click chemistry. MinION sequencing of yeast and human poly(A) mRNAs revealed high-confidence full-length reads with improved TSS coverage and allowed identification of alternative isoforms.

Identifying full-length transcripts in eukaryotic mRNA using nanopore sequencing

ACS Spring Meeting 2021

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