biostudies-literatureUnknownAkeson SNHGRI NIH HHSNIGMS NIH HHSWe describe application of Nanopore direct tRNA sequencing and ionic current signal for isodecoder-level analysis of bacterial and yeast tRNA. The computational advance is the application of deep learning for predicting tRNA isodecoders directly from raw nanopore ionic current signals. We then combined ionic current analysis with a pairwise sequence alignment strategy to improve tRNA sequence alignments. We applied this strategy to Nanopore data for <i>E. coli</i> and <i>S. cerevisiae</i> tRNA and aligned 2.6% and 13.1% more reads respectively when compared to present strategies for Nanopore tRNA analysis. Notably, this increase in alignments also came with a significant increase in alignment identity. The ionic current models confirmed enrichment of both an individual tRNA isotype and a tbioRxiv : the preprint server for biology2025.12.27.696718https://www.ebi.ac.uk/biostudies/studies/S-EPMC12767535biostudies-literaturetRNA isodecoder analysis using Nanopore ionic current signals and deep learning.PMC12767535R01 HG013876T32 GM149387R35 GM143125Garcia DMAkeson SBarry MLKoutmou KSJain MEsfahani NGKakhaki PDAbu-Shumays RLTzadikario TReinsch JLZamecnik MfalsetRNA isodecoder analysis using Nanopore ionic current signals and deep learning.We describe application of Nanopore direct tRNA sequencing and ionic current signal for isodecoder-level analysis of bacterial and yeast tRNA. The computational advance is the application of deep learning for predicting tRNA isodecoders directly from raw nanopore ionic current signals. We then combined ionic current analysis with a pairwise sequence alignment strategy to improve tRNA sequence alignments. We applied this strategy to Nanopore data for <i>E. coli</i> and <i>S. cerevisiae</i> tRNA and aligned 2.6% and 13.1% more reads respectively when compared to present strategies for Nanopore tRNA analysis. Notably, this increase in alignments also came with a significant increase in alignment identity. The ionic current models confirmed enrichment of both an individual tRNA isotype and a t2025-01-01T00:00:00Z2025 Dec2026-06-04T03:21:34.753Z2026-06-04T03:12:34.032ZS-EPMC127675354149762910.64898/2025.12.27.696718