Direct Nanopore Sequencing of Individual Full Length tRNA Strands.
basic_science · Level V
Where this comes from
- Record sourced from PubMed, PMID 34618430.
- Also identified by DOI 10.1021/acsnano.1c06488 and PMC identifier 10189790.
- Licence recorded as CC BY-NC-ND.
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Abstract
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 <i>E. coli</i> tRNA<sup>fMet</sup>, tRNA<sup>Lys</sup>, and tRNA<sup>Phe</sup> 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 <i>E. coli</i> 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 <i>E. coli</i> 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.
Medical subject headings
- Nanopore Sequencing
- Nanopores