{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,18]],"date-time":"2026-09-18T05:06:25Z","timestamp":1789707985113,"version":"4.0.1"},"reference-count":41,"publisher":"Wiley","license":[{"start":{"date-parts":[[2026,9,17]],"date-time":"2026-09-17T00:00:00Z","timestamp":1789603200000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by\/4.0\/"},{"start":{"date-parts":[[2026,9,17]],"date-time":"2026-09-17T00:00:00Z","timestamp":1789603200000},"content-version":"tdm","delay-in-days":0,"URL":"http:\/\/doi.wiley.com\/10.1002\/tdm_license_1.1"}],"funder":[{"DOI":"10.13039\/501100001809","name":"National Natural Science Foundation of China","doi-asserted-by":"publisher","award":["82173046"],"award-info":[{"award-number":["82173046"]}],"id":[{"id":"10.13039\/501100001809","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100001809","name":"National Natural Science Foundation of China","doi-asserted-by":"publisher","award":["82472702"],"award-info":[{"award-number":["82472702"]}],"id":[{"id":"10.13039\/501100001809","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":["advanced.onlinelibrary.wiley.com"],"crossmark-restriction":true},"short-container-title":["Advanced Science"],"abstract":"<jats:title>ABSTRACT<\/jats:title>\n                  <jats:p>Differential transcript usage (DTU) analysis reveals transcript\u2010level regulation in alternative splicing. With the rapid adoption of long\u2010read sequencing in bulk, single\u2010cell, and spatial transcriptomics, reliable evaluation of DTU methods under real biological conditions becomes essential. Current evaluation frameworks mainly rely on simulated data, which can introduce bias and may not reflect true regulatory mechanisms. A real\u2010data\u2010driven framework is constructed to evaluate DTU methods across long\u2010read bulk, single\u2010cell, and spatial transcriptomics. The framework includes two key components. First, a biologically grounded reference transcript set is defined using RNA\u2010binding protein (RBP) knockout or knockdown RNA\u2010seq data together with experimentally validated RBP\u2010transcript interactions. Second, a DTU\u2010specific evaluation metric, the transcript set enrichment score, is introduced to quantify how effectively a method prioritizes reference transcripts in ranked results. The framework is systematically validated for reliability, unbiasedness, stability, effectiveness, and robustness using multiple real RNA\u2010seq datasets. Supported by this validation, ten representative DTU methods are evaluated across long\u2010read and short\u2010read data, revealing performance differences across data types. Beyond evaluating DTU methods, the framework is further extended to predict transcript\u2010level RBP activity, recovering perturbed RBPs more consistently than gene\u2010level differential expression strategies. Together, this study establishes a biologically interpretable and data\u2010driven standard for DTU method evaluation.<\/jats:p>","DOI":"10.1002\/advs.77756","type":"journal-article","created":{"date-parts":[[2026,9,18]],"date-time":"2026-09-18T04:37:08Z","timestamp":1789706228000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["A Real\u2010Data\u2010Driven Framework for Evaluating Differential Transcript Usage Methods Across Long\u2010Read Bulk, Single\u2010Cell, and Spatial Transcriptomics"],"prefix":"10.1002","author":[{"given":"Chenxing","family":"Zhang","sequence":"first","affiliation":[{"name":"State Key Laboratory of Holistic Integrative Management of Gastrointestinal Cancers and Department of Immunology Fourth Military Medical University  Xi'an Shaanxi China"},{"name":"State Key Laboratory of Holistic Integrative Management of Gastrointestinal Cancers and Department of Biochemistry and Molecular Biology Fourth Military Medical University  Xi'an Shaanxi China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jun","family":"Liu","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Holistic Integrative Management of Gastrointestinal Cancers and Department of Immunology Fourth Military Medical University  Xi'an Shaanxi China"},{"name":"State Key Laboratory of Holistic Integrative Management of Gastrointestinal Cancers and Department of Biochemistry and Molecular Biology Fourth Military Medical University  Xi'an Shaanxi China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Qi","family":"Zhao","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Holistic Integrative Management of Gastrointestinal Cancers and Department of Immunology Fourth Military Medical University  Xi'an Shaanxi China"},{"name":"State Key Laboratory of Holistic Integrative Management of Gastrointestinal Cancers and Department of Biochemistry and Molecular Biology Fourth Military Medical University  Xi'an Shaanxi China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Huilong","family":"Yin","sequence":"additional","affiliation":[{"name":"Henan Collaborative Innovation Center of Molecular Diagnosis and Laboratory Medicine School of Medical Technology Xinxiang Medical University  Xinxiang Henan China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-8854-4672","authenticated-orcid":false,"given":"Angang","family":"Yang","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Holistic Integrative Management of Gastrointestinal Cancers and Department of Immunology Fourth Military Medical University  Xi'an Shaanxi China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Minhua","family":"Zheng","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Holistic Integrative Management of Gastrointestinal Cancers and Department of Medical Genetics and Developmental Biology Fourth Military Medical University  Xi'an Shaanxi China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-6147-1573","authenticated-orcid":false,"given":"Rui","family":"Zhang","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Holistic Integrative Management of Gastrointestinal Cancers and Department of Immunology Fourth Military Medical University  Xi'an Shaanxi China"},{"name":"State Key Laboratory of Holistic Integrative Management of Gastrointestinal Cancers and Department of Biochemistry and Molecular Biology Fourth Military Medical University  Xi'an Shaanxi China"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"311","published-online":{"date-parts":[[2026,9,17]]},"reference":[{"key":"e_1_2_11_2_1","doi-asserted-by":"crossref","first-page":"1521","DOI":"10.12688\/f1000research.7563.2","article-title":"Differential Analyses for RNA\u2010seq: Transcript\u2010Level Estimates Improve Gene\u2010Level Inferences","volume":"4","author":"Soneson C.","year":"2016","journal-title":"F1000Research"},{"issue":"1","key":"e_1_2_11_3_1","doi-asserted-by":"crossref","first-page":"12","DOI":"10.1186\/s13059-015-0862-3","article-title":"Isoform Prefiltering Improves Performance of Count\u2010Based Methods for Analysis of Differential Transcript Usage","volume":"17","author":"Soneson C.","year":"2016","journal-title":"Genome Biology"},{"issue":"7221","key":"e_1_2_11_4_1","doi-asserted-by":"crossref","first-page":"470","DOI":"10.1038\/nature07509","article-title":"Alternative Isoform Regulation in Human Tissue Transcriptomes","volume":"456","author":"Wang E. 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