posted on 2021-10-07, 17:39authored byNiki K. Thomas, Vinay C. Poodari, Miten Jain, Hugh E. Olsen, Mark Akeson, Robin L. Abu-Shumays
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 transfer RNA (tRNA)
molecules at subnanometer 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. 76–92% 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 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.