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Here, we present a comprehensive benchmark of seven AS detection tools\u2014replicate Multivariate Analysis of Transcript Splicing (rMATS), Super-fast Pipeline for alternative splicing analysis, version 2 (SUPPA2), Modeling Alternative Junction Inclusion Quantification + Visualization Of Inferred Local splicing Alternatives (MAJIQ+VOILA), EventPointerST (EPST), Shiba, LeafCutter, and the newly introduced EventPointer BAM (EPBAM)\u2014evaluated across three complementary datasets: reverse transcription PCR (RT-PCR) validated experiments, simulated data, and Lexogen Spike-In RNA Variant (SIRV). The inclusion of SIRV, which provide experimentally derived sequencing data with fully controlled ground truth, is central to our evaluation framework, enabling rigorous assessment of quantification accuracy under real technical conditions. We assess tool performance under three annotation scenarios\u2014complete, incomplete, and overloaded\u2014reflecting the annotation uncertainty commonly encountered in practice. EPBAM extends the EventPointer framework by enabling de novo event detection coupled with a coverage-corrected \u03a8 quantification strategy. Our results demonstrate that annotation quality substantially drives tool performance: annotation-dependent tools achieve peak accuracy under complete annotations, whereas EPBAM exhibits the most robust and consistent behaviour across annotation conditions, recovering unannotated events with low false discovery rates. No single tool dominates across all evaluation metrics, highlighting the importance of dataset diversity and standardized benchmarking practices for meaningful tool comparison in the AS field.<\/jats:p>","DOI":"10.1093\/bib\/bbag556","type":"journal-article","created":{"date-parts":[[2026,9,22]],"date-time":"2026-09-22T11:41:49Z","timestamp":1790077309000},"source":"Crossref","is-referenced-by-count":0,"title":["EPBAM: annotation-robust detection of alternative splicing events and a systematic benchmarking of current tools"],"prefix":"10.1093","volume":"27","author":[{"given":"Cesar","family":"Lobato-Fernandez","sequence":"first","affiliation":[{"name":"Biomedical Engineering and Science Department, TECNUN, University of Navarra, Paseo de Manuel Lardiz\u00e1bal 13 , 20018 Donostia-San Sebasti\u00e1n, Gipuzkoa ,","place":["Spain"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0003-2860-2222","authenticated-orcid":false,"given":"Laura","family":"Jareno","sequence":"additional","affiliation":[{"name":"Biomedical Engineering and Science Department, TECNUN, University of Navarra, Paseo de Manuel Lardiz\u00e1bal 13 , 20018 Donostia-San Sebasti\u00e1n, Gipuzkoa ,","place":["Spain"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-3274-2450","authenticated-orcid":false,"given":"Angel","family":"Rubio","sequence":"additional","affiliation":[{"name":"Biomedical Engineering and Science Department, TECNUN, University of Navarra, Paseo de Manuel Lardiz\u00e1bal 13 , 20018 Donostia-San Sebasti\u00e1n, Gipuzkoa ,","place":["Spain"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-1588-2195","authenticated-orcid":false,"given":"Juan 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