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Direct Nanopore Sequencing of Individual Full Length tRNA Strands

We describe a method for direct tRNA sequencing using the Oxford Nanopore MinION. The principal technical advance is custom adapters that facilitate end-to-end sequencing of individual tRNA molecules. A second advance is a Nanopore sequencing pipeline optimized for tRNA. We tested this method using purified E. coli tRNAfMet, tRNALys, and tRNAPhe samples. 76-to-92% of individual aligned tRNA sequence reads were full length. As proof of concept, we showed that Nanopore sequencing detected all 42 expected tRNA isoacceptors in total E. coli tRNA. Alignment-based comparison between the three purified tRNAs and their synthetic controls revealed systematic miscalls at or adjacent to the positions of known nucleotide modifications. Systematic miscalls were also observed proximal to known modifications in total E. coli tRNA alignments.