Nanopore direct RNA sequencing and the epitranscriptome: Advances in mapping native RNA landscapes

@article{zhang2026nanopore,
  title = {{Nanopore direct RNA sequencing and the epitranscriptome: Advances in mapping native RNA landscapes}},
  author = {Tianyuan Zhang and Jia Li and Chao Tang and You Wu and H Wu and Zhu Xi-Tong and Ziyang Luo and Hang Qin and Lishan Ding and Yu Zeng and Shiou Yih Lee and Xiaotao Shen and Shiwen Gao and Zhaoyang Tian and Qian Tang and Mian Li and Muhammad Tahir ul Qamar and Yang Dong and Komivi Dossa and Yaxuan Zhang and Hu Chen and Sanqi An and Xiang Yu and Lu Chen and Dingjie Wang and Shengli Li and Ling‐Ling Chen and Yanqiang Li},
  journal = {iMeta},
  year = {2026},
  month = jun,
  doi = {10.1002/imt2.70136},
  url = {https://doi.org/10.1002/imt2.70136},
  note = {CC BY},
}

TY  - JOUR
TI  - Nanopore direct RNA sequencing and the epitranscriptome: Advances in mapping native RNA landscapes
AU  - Tianyuan Zhang
AU  - Jia Li
AU  - Chao Tang
AU  - You Wu
AU  - H Wu
AU  - Zhu Xi-Tong
AU  - Ziyang Luo
AU  - Hang Qin
AU  - Lishan Ding
AU  - Yu Zeng
AU  - Shiou Yih Lee
AU  - Xiaotao Shen
AU  - Shiwen Gao
AU  - Zhaoyang Tian
AU  - Qian Tang
AU  - Mian Li
AU  - Muhammad Tahir ul Qamar
AU  - Yang Dong
AU  - Komivi Dossa
AU  - Yaxuan Zhang
AU  - Hu Chen
AU  - Sanqi An
AU  - Xiang Yu
AU  - Lu Chen
AU  - Dingjie Wang
AU  - Shengli Li
AU  - Ling‐Ling Chen
AU  - Yanqiang Li
PY  - 2026
DA  - 2026/06/01
JO  - iMeta
DO  - 10.1002/imt2.70136
UR  - https://doi.org/10.1002/imt2.70136
AB  - Nanopore direct RNA sequencing (DRS) has transformed transcriptomics by enabling single-molecule, long-read sequencing of native RNA without the need for reverse transcription or amplification. In contrast to short-read RNA-seq and cDNA-based long-read approaches, DRS can simultaneously capture multiple RNA modifications, full-length transcript architecture, alternative splicing patterns, and poly(A) tail features within individual molecules, thereby providing an integrated view of transcriptomic and epitranscriptomic regulation. In this comprehensive review, we outline the biophysical principles underlying nanopore DRS and trace its technological evolution. We compare its performance with short-read RNA sequencing, long-read cDNA sequencing, and conventional RNA-modification mapping strategies, highlighting its advantages in isoform-resolved quantification and multilayer RNA feature integration, while also clarifying contexts in which alternative or combined approaches may be more appropriate for robust biological interpretation. We further summarize optimized experimental workflows, including library construction strategies tailored to diverse RNA biotypes (mRNA, rRNA, tRNA, circRNA, miRNA, and nonpoly(A) transcripts), as well as recommended quality-control procedures and sequencing optimization practices. Emphasizing recent computational advances and translational applications of DRS, we cover state-of-the-art algorithms for RNA modification detection, transcript reconstruction, and isoform quantification. We also propose analytical pipelines for poly(A) tail length inference and integrative frameworks that jointly analyze these regulatory layers. We distinguish direct nanopore signals from computational inferences to define confidence levels and emphasize benchmarking and orthogonal validation of readouts. Practical implementation examples are included to facilitate reproducible analysis. Finally, we highlight emerging applications of integrated DRS, including the resolution of complex transcriptomes, the characterization of coordinated epitranscriptomic regulation, and the identification of disease-associated RNA signatures. We also discuss current technical challenges and future perspectives, particularly in relation to multi-omics integration and the broader deployment of DRS in precision medicine as well as in plant and animal research.
SN  - 2770-596X
SN  - 2770-5986
ER  -