PubReading [156] - Direct Nanopore Sequencing of Individual Full Length tRNA Strands - N. Thomas, R. Abu-Shumays et al

<p>We describe a method for direct tRNA <strong>sequencing</strong> using the <strong>Oxford Nanopore MinION.</strong> The principal technical advance is custom adapters that facilitate end-to-end sequencing of individual <strong>transfer RNA</strong> (tRNA) molecules at subnanometer precision. A second advance is a nanopore sequencing pipeline optimized for tRNA. We tested this method using purified <em>E. coli</em> tRNAfMet, tRNALys, and tRNAPhe samples. 76–92% of individual aligned tRNA sequence reads were full length. As a proof of concept, we showed that nanopore sequencing detected all 43 expected isoacceptors in total <em>E. coli</em> MRE600 tRNA as well as isodecoders that further define that tRNA population. Alignment-based comparisons between the three purified <strong>tRNA</strong>s and their synthetic controls revealed systematic nucleotide miscalls that were diagnostic of known modifications. Systematic miscalls were also observed proximal to known modifications in total <em>E. coli</em> tRNA alignments, including a highly conserved pseudouridine in the T loop. This work highlights the potential of nanopore direct tRNA sequencing as well as improvements needed to implement tRNA sequencing for human healthcare applications.</p><p><em>doi.org/10.1021/acsnano.1c06488 - 2021</em></p>