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In contrast to short\u2010read RNA\u2010seq and cDNA\u2010based long\u2010read approaches, DRS can simultaneously capture multiple RNA modifications, full\u2010length 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\u2010read RNA sequencing, long\u2010read cDNA sequencing, and conventional RNA\u2010modification mapping strategies, highlighting its advantages in isoform\u2010resolved 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\u2010control procedures and sequencing optimization practices. Emphasizing recent computational advances and translational applications of DRS, we cover state\u2010of\u2010the\u2010art 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\u2010associated RNA signatures. We also discuss current technical challenges and future perspectives, particularly in relation to multi\u2010omics integration and the broader deployment of DRS in precision medicine as well as in plant and animal research.<\/jats:p>","DOI":"10.1002\/imt2.70136","type":"journal-article","created":{"date-parts":[[2026,7,1]],"date-time":"2026-07-01T20:23:11Z","timestamp":1782937391000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":9,"title":["Nanopore direct RNA sequencing and the epitranscriptome: Advances in mapping native RNA landscapes"],"prefix":"10.1002","volume":"5","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-8968-563X","authenticated-orcid":false,"given":"Tianyuan","family":"Zhang","sequence":"first","affiliation":[{"name":"Department of Urology The First Affiliated Hospital of Xi'an Jiaotong University Xi'an China"},{"name":"Benagen Institute Wuhan 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