A direct RNA sequencing workflow for simultaneous profiling of tRNA abundance and modifications in mouse liver

Abstract

Transfer RNA (tRNA) is highly modified and difficult to profile using reverse-transcription-based sequencing. Here, we established a direct RNA sequencing (tRNA-DRS) workflow to simultaneously characterize tRNA abundance and modification signals in mouse liver. The workflow included small-RNA enrichment, deacylation, splint-adapter ligation, barcoding, reverse transcription, and nanopore sequencing on PromethION with modification-aware basecalling. From 1,678,666 raw reads, 1,421,279 (84.67%) reads were retained after 20-200 nt length filtering, and 17,094 uniquely mapped reads (MAPQ [≥] 10) were obtained. These reads covered 244 tRNA genes and represented all 23 tRNA isotypes. tRNA abundance was strongly skewed, with Asp, Gly, Ser, and Lys accounting for 56.6% of uniquely mapped reads. Read-body coverage indicated that most uniquely mapped reads spanned substantial portions of tRNA molecules (mean 71.1%, median 75.9%; 83.3% reads covering [≥]50% of the reference). Modification analysis identified 41,013 candidate sites, of which 711 passed stringent filtering (modification fraction [≥]50%, coverage [≥]5), spanning eight modification types and dominated by pseudouridine and m5C. Filtered sites showed position-specific clustering and isotype-dependent patterns. Together, these results support tRNA-DRS as a practical single-assay framework for simultaneous tRNA expression and epitranscriptomic profiling in mammalian tissue.

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Cite

BibTeX RIS

BibTeX

@misc{dai2026direct,
  title = {{A direct RNA sequencing workflow for simultaneous profiling of tRNA abundance and modifications in mouse liver}},
  author = {S. Dai and Z. Tian and Y. Zhang and X. Li and X. Yi and T. Qiu and L. Yang and J. Ke and M. Chen and J. Huang and X. Hu and Y. Li and H. Chen and T. Zhang},
  howpublished = {bioRxiv},
  year = {2026},
  month = oct,
  doi = {10.64898/2026.10.04.754913},
  url = {https://www.biorxiv.org/content/10.64898/2026.10.04.754913v1},
  note = {CC BY-NC-ND},
}

RIS

TY  - UNPB
TI  - A direct RNA sequencing workflow for simultaneous profiling of tRNA abundance and modifications in mouse liver
AU  - S. Dai
AU  - Z. Tian
AU  - Y. Zhang
AU  - X. Li
AU  - X. Yi
AU  - T. Qiu
AU  - L. Yang
AU  - J. Ke
AU  - M. Chen
AU  - J. Huang
AU  - X. Hu
AU  - Y. Li
AU  - H. Chen
AU  - T. Zhang
PY  - 2026
DA  - 2026/10/04
PB  - bioRxiv
DO  - 10.64898/2026.10.04.754913
UR  - https://www.biorxiv.org/content/10.64898/2026.10.04.754913v1
AB  - Transfer RNA (tRNA) is highly modified and difficult to profile using reverse-transcription-based sequencing. Here, we established a direct RNA sequencing (tRNA-DRS) workflow to simultaneously characterize tRNA abundance and modification signals in mouse liver. The workflow included small-RNA enrichment, deacylation, splint-adapter ligation, barcoding, reverse transcription, and nanopore sequencing on PromethION with modification-aware basecalling. From 1,678,666 raw reads, 1,421,279 (84.67%) reads were retained after 20-200 nt length filtering, and 17,094 uniquely mapped reads (MAPQ [≥] 10) were obtained. These reads covered 244 tRNA genes and represented all 23 tRNA isotypes. tRNA abundance was strongly skewed, with Asp, Gly, Ser, and Lys accounting for 56.6% of uniquely mapped reads. Read-body coverage indicated that most uniquely mapped reads spanned substantial portions of tRNA molecules (mean 71.1%, median 75.9%; 83.3% reads covering [≥]50% of the reference). Modification analysis identified 41,013 candidate sites, of which 711 passed stringent filtering (modification fraction [≥]50%, coverage [≥]5), spanning eight modification types and dominated by pseudouridine and m5C. Filtered sites showed position-specific clustering and isotype-dependent patterns. Together, these results support tRNA-DRS as a practical single-assay framework for simultaneous tRNA expression and epitranscriptomic profiling in mammalian tissue.
ER  -

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