ena-STUDY-UC SANTA CRUZ-16-09-2021-17:41:51:990-301
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 at sub-nanometer precision. 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 43 expected isoacceptors in total E. coli MRE600 tRNA, as well as isodecoders that further define that tRNA population. Alignment-based comparisons between the three purified tRNAs 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 E. coli 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.
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 at sub-nanometer precision. 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 43 expected isoacceptors in total E. coli MRE600 tRNA, as well as isodecoders that further define that tRNA population. Alignment-based comparisons between the three purified tRNAs 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 E. coli 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.