{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,8,18]],"date-time":"2026-08-18T04:49:19Z","timestamp":1787028559131,"version":"build-2736575974"},"reference-count":43,"publisher":"MDPI AG","issue":"7","license":[{"start":{"date-parts":[[2025,3,30]],"date-time":"2025-03-30T00:00:00Z","timestamp":1743292800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100001691","name":"Japan Society for the Promotion of Science (JSPS)","doi-asserted-by":"publisher","award":["12J09944"],"award-info":[{"award-number":["12J09944"]}],"id":[{"id":"10.13039\/501100001691","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100001691","name":"Japan Society for the Promotion of Science (JSPS)","doi-asserted-by":"publisher","award":["22K07854"],"award-info":[{"award-number":["22K07854"]}],"id":[{"id":"10.13039\/501100001691","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100001691","name":"Japan Society for the Promotion of Science (JSPS)","doi-asserted-by":"publisher","award":["23K05607"],"award-info":[{"award-number":["23K05607"]}],"id":[{"id":"10.13039\/501100001691","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100001691","name":"Japan Society for the Promotion of Science (JSPS)","doi-asserted-by":"publisher","award":["10955"],"award-info":[{"award-number":["10955"]}],"id":[{"id":"10.13039\/501100001691","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100001691","name":"Japan Society for the Promotion of Science (JSPS)","doi-asserted-by":"publisher","award":["11181"],"award-info":[{"award-number":["11181"]}],"id":[{"id":"10.13039\/501100001691","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100001691","name":"Japan Society for the Promotion of Science (JSPS)","doi-asserted-by":"publisher","award":["26699"],"award-info":[{"award-number":["26699"]}],"id":[{"id":"10.13039\/501100001691","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100001691","name":"Japan Society for the Promotion of Science (JSPS)","doi-asserted-by":"publisher","award":["10663"],"award-info":[{"award-number":["10663"]}],"id":[{"id":"10.13039\/501100001691","id-type":"DOI","asserted-by":"publisher"}]},{"name":"Tokumori Yasumoto Memorial Trust for Researchers on Tuberous Sclerosis Complex and Related Rare Neurological Disease","award":["12J09944"],"award-info":[{"award-number":["12J09944"]}]},{"name":"Tokumori Yasumoto Memorial Trust for Researchers on Tuberous Sclerosis Complex and Related Rare Neurological Disease","award":["22K07854"],"award-info":[{"award-number":["22K07854"]}]},{"name":"Tokumori Yasumoto Memorial Trust for Researchers on Tuberous Sclerosis Complex and Related Rare Neurological Disease","award":["23K05607"],"award-info":[{"award-number":["23K05607"]}]},{"name":"Tokumori Yasumoto Memorial Trust for Researchers on Tuberous Sclerosis Complex and Related Rare Neurological Disease","award":["10955"],"award-info":[{"award-number":["10955"]}]},{"name":"Tokumori Yasumoto Memorial Trust for Researchers on Tuberous Sclerosis Complex and Related Rare Neurological Disease","award":["11181"],"award-info":[{"award-number":["11181"]}]},{"name":"Tokumori Yasumoto Memorial Trust for Researchers on Tuberous Sclerosis Complex and Related Rare Neurological Disease","award":["26699"],"award-info":[{"award-number":["26699"]}]},{"name":"Tokumori Yasumoto Memorial Trust for Researchers on Tuberous Sclerosis Complex and Related Rare Neurological Disease","award":["10663"],"award-info":[{"award-number":["10663"]}]},{"DOI":"10.13039\/501100004043","name":"Kanazawa Medical University","doi-asserted-by":"publisher","award":["12J09944"],"award-info":[{"award-number":["12J09944"]}],"id":[{"id":"10.13039\/501100004043","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100004043","name":"Kanazawa Medical University","doi-asserted-by":"publisher","award":["22K07854"],"award-info":[{"award-number":["22K07854"]}],"id":[{"id":"10.13039\/501100004043","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100004043","name":"Kanazawa Medical University","doi-asserted-by":"publisher","award":["23K05607"],"award-info":[{"award-number":["23K05607"]}],"id":[{"id":"10.13039\/501100004043","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100004043","name":"Kanazawa Medical University","doi-asserted-by":"publisher","award":["10955"],"award-info":[{"award-number":["10955"]}],"id":[{"id":"10.13039\/501100004043","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100004043","name":"Kanazawa Medical University","doi-asserted-by":"publisher","award":["11181"],"award-info":[{"award-number":["11181"]}],"id":[{"id":"10.13039\/501100004043","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100004043","name":"Kanazawa Medical University","doi-asserted-by":"publisher","award":["26699"],"award-info":[{"award-number":["26699"]}],"id":[{"id":"10.13039\/501100004043","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100004043","name":"Kanazawa Medical University","doi-asserted-by":"publisher","award":["10663"],"award-info":[{"award-number":["10663"]}],"id":[{"id":"10.13039\/501100004043","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["IJMS"],"abstract":"<jats:p>RNA sequencing (RNA-Seq) is a powerful technique for the quantification of transcripts and the analysis of alternative splicing. Previously, our laboratory developed the targeted RNA long-amplicon sequencing (rLAS) method, which has the advantage of allowing deep analysis of targeted specific transcripts. The computational tools for analyzing RNA-Seq data have boosted alternative splicing research by detecting and quantifying splicing events. However, the performance of these splicing tools has not yet been investigated for rLAS. Here, we evaluated the performance of four splicing tools (MAJIQ, rMATS, MISO, and SplAdder) using samples with different types of known splicing events (exon-skipping, multiple-exon-skipping, alternative 5\u2032 splicing, and alternative 3\u2032 splicing). MAJIQ was able to detect all of the types of events, but it was unable to detect one of the exon-skipping events. On the other hand, rMATS was able to detect all of the exon-skipping events. However, rMATS failed to detect other types of events besides exon-skipping events. Both MISO and SplAdder were unable to detect any of the events. These results indicate that MAJIQ presents better performance for the different types of splicing events in rLAS and that rMATS shows better performance for exon-skipping splicing events.<\/jats:p>","DOI":"10.3390\/ijms26073220","type":"journal-article","created":{"date-parts":[[2025,3,31]],"date-time":"2025-03-31T03:25:02Z","timestamp":1743391502000},"page":"3220","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":1,"title":["Computational Comparison of Differential Splicing Tools for Targeted RNA Long-Amplicon Sequencing (rLAS)"],"prefix":"10.3390","volume":"26","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-7600-3481","authenticated-orcid":false,"given":"Hiroki","family":"Ura","sequence":"first","affiliation":[{"name":"Center for Clinical Genomics, Kanazawa Medical University Hospital, 1-1 Daigaku, Uchinada, Kahoku 920-0923, Ishikawa, Japan"},{"name":"Division of Genomic Medicine, Department of Advanced Medicine, Medical Research Institute, Kanazawa Medical University, 1-1 Daigaku, Uchinada, Kahoku 920-0923, Ishikawa, Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Hisayo","family":"Hatanaka","sequence":"additional","affiliation":[{"name":"Center for Clinical Genomics, Kanazawa Medical University Hospital, 1-1 Daigaku, Uchinada, Kahoku 920-0923, Ishikawa, Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-3189-8111","authenticated-orcid":false,"given":"Sumihito","family":"Togi","sequence":"additional","affiliation":[{"name":"Center for Clinical Genomics, Kanazawa Medical University Hospital, 1-1 Daigaku, Uchinada, Kahoku 920-0923, Ishikawa, Japan"},{"name":"Division of Genomic Medicine, Department of Advanced Medicine, Medical Research Institute, Kanazawa Medical University, 1-1 Daigaku, Uchinada, Kahoku 920-0923, Ishikawa, Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-5641-7962","authenticated-orcid":false,"given":"Yo","family":"Niida","sequence":"additional","affiliation":[{"name":"Center for Clinical Genomics, Kanazawa Medical University Hospital, 1-1 Daigaku, Uchinada, Kahoku 920-0923, Ishikawa, Japan"},{"name":"Division of Genomic Medicine, Department of Advanced Medicine, Medical Research Institute, Kanazawa Medical University, 1-1 Daigaku, Uchinada, Kahoku 920-0923, Ishikawa, Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2025,3,30]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"257","DOI":"10.1038\/nrg.2016.10","article-title":"Translating RNA sequencing into clinical diagnostics: Opportunities and challenges","volume":"17","author":"Byron","year":"2016","journal-title":"Nat. Rev. Genet."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"621","DOI":"10.1038\/nmeth.1226","article-title":"Mapping and quantifying mammalian transcriptomes by RNA-Seq","volume":"5","author":"Mortazavi","year":"2008","journal-title":"Nat. Methods"},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"956","DOI":"10.1126\/science.1160342","article-title":"A global view of gene activity and alternative splicing by deep sequencing of the human transcriptome","volume":"321","author":"Sultan","year":"2008","journal-title":"Science"},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"660","DOI":"10.1126\/science.aaa0355","article-title":"Human genomics. The human transcriptome across tissues and individuals","volume":"348","author":"Ferreira","year":"2015","journal-title":"Science"},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"75","DOI":"10.1038\/nrg2673","article-title":"RNA processing and its regulation: Global insights into biological networks","volume":"11","author":"Licatalosi","year":"2010","journal-title":"Nat. Rev. Genet."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"470","DOI":"10.1038\/nature07509","article-title":"Alternative isoform regulation in human tissue transcriptomes","volume":"456","author":"Wang","year":"2008","journal-title":"Nature"},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"1413","DOI":"10.1038\/ng.259","article-title":"Deep surveying of alternative splicing complexity in the human transcriptome by high-throughput sequencing","volume":"40","author":"Pan","year":"2008","journal-title":"Nat. Genet."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"355","DOI":"10.1042\/CS20160211","article-title":"RNA splicing in human disease and in the clinic","volume":"131","author":"Baralle","year":"2017","journal-title":"Clin. Sci."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"152","DOI":"10.3892\/br.2014.407","article-title":"Mechanism of alternative splicing and its regulation","volume":"3","author":"Wang","year":"2015","journal-title":"Biomed. Rep."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"1029","DOI":"10.1261\/rna.032243.112","article-title":"Alternative splicing networks regulated by signaling in human T cells","volume":"18","author":"Martinez","year":"2012","journal-title":"RNA"},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"8768","DOI":"10.1038\/ncomms9768","article-title":"ESRP2 controls an adult splicing programme in hepatocytes to support postnatal liver maturation","volume":"6","author":"Bhate","year":"2015","journal-title":"Nat. Commun."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"8933","DOI":"10.1093\/nar\/gkw560","article-title":"The alternative splicing program of differentiated smooth muscle cells involves concerted non-productive splicing of post-transcriptional regulators","volume":"44","author":"Llorian","year":"2016","journal-title":"Nucleic Acids Res."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"3603","DOI":"10.1038\/ncomms4603","article-title":"Alternative splicing regulates vesicular trafficking genes in cardiomyocytes during postnatal heart development","volume":"5","author":"Giudice","year":"2014","journal-title":"Nat. Commun."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"499","DOI":"10.1038\/nn.3332","article-title":"mRNA expression, splicing and editing in the embryonic and adult mouse cerebral cortex","volume":"16","author":"Dillman","year":"2013","journal-title":"Nat. Neurosci."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"584","DOI":"10.1016\/j.clinbiochem.2004.05.015","article-title":"Splice variants as cancer biomarkers","volume":"37","author":"Brinkman","year":"2004","journal-title":"Clin. Biochem."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"2635","DOI":"10.1242\/jcs.03053","article-title":"The connection between splicing and cancer","volume":"119","author":"Srebrow","year":"2006","journal-title":"J. Cell Sci."},{"key":"ref_17","doi-asserted-by":"crossref","unstructured":"Ura, H., and Niida, Y. (2024). Comparison of RNA-Sequencing Methods for Degraded RNA. Int. J. Mol. Sci., 25.","DOI":"10.3390\/ijms25116143"},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"424","DOI":"10.1016\/j.jmoldx.2020.12.009","article-title":"Optimization and Validation of Multimodular, Long-Range PCR-Based Next-Generation Sequencing Assays for Comprehensive Detection of Mutation in Tuberous Sclerosis Complex","volume":"23","author":"Togi","year":"2021","journal-title":"J. Mol. Diagn. JMD"},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"357","DOI":"10.1007\/978-1-62703-980-2_26","article-title":"Methods to study splicing from high-throughput RNA sequencing data","volume":"1126","author":"Alamancos","year":"2014","journal-title":"Methods Mol. Biol."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"1230","DOI":"10.1038\/s41467-023-36585-y","article-title":"RNA splicing analysis using heterogeneous and large RNA-seq datasets","volume":"14","author":"Aicher","year":"2023","journal-title":"Nat. Commun."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"1083","DOI":"10.1038\/s41596-023-00944-2","article-title":"rMATS-turbo: An efficient and flexible computational tool for alternative splicing analysis of large-scale RNA-seq data","volume":"19","author":"Wang","year":"2024","journal-title":"Nat. Protoc."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"1009","DOI":"10.1038\/nmeth.1528","article-title":"Analysis and design of RNA sequencing experiments for identifying isoform regulation","volume":"7","author":"Katz","year":"2010","journal-title":"Nat. Methods"},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"1840","DOI":"10.1093\/bioinformatics\/btw076","article-title":"SplAdder: Identification, quantification and testing of alternative splicing events from RNA-Seq data","volume":"32","author":"Kahles","year":"2016","journal-title":"Bioinformatics"},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"102926","DOI":"10.1016\/j.scr.2022.102926","article-title":"Establishment of a human induced pluripotent stem cell line, KMUGMCi005-A, from a patient with Epidermodysplasia verruciformis (EV) bearing homozygous splicing donor site mutation in the TMC8 gene","volume":"64","author":"Ura","year":"2022","journal-title":"Stem. Cell. Res."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"103388","DOI":"10.1016\/j.scr.2024.103388","article-title":"Establishment of a human induced pluripotent stem cell line, KMUGMCi010-A, from a patient with X-linked Ohdo syndrome bearing missense mutation in the MED12 gene","volume":"77","author":"Ura","year":"2024","journal-title":"Stem Cell Res."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"e63628","DOI":"10.1002\/ajmg.a.63628","article-title":"Qualitative and quantitative analysis of MED12 c.887G>A causing both missense and splicing variants in X-linked Ohdo syndrome","volume":"194","author":"Togi","year":"2024","journal-title":"Am. J. Med. Genet. A"},{"key":"ref_27","doi-asserted-by":"crossref","unstructured":"Simoneau, J., Gosselin, R., and Scott, M.S. (2020). Factorial study of the RNA-seq computational workflow identifies biases as technical gene signatures. NAR Genom. Bioinform., 2.","DOI":"10.1093\/nargab\/lqaa043"},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"357","DOI":"10.1038\/nmeth.3317","article-title":"HISAT: A fast spliced aligner with low memory requirements","volume":"12","author":"Kim","year":"2015","journal-title":"Nat. Methods"},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"15","DOI":"10.1093\/bioinformatics\/bts635","article-title":"STAR: Ultrafast universal RNA-seq aligner","volume":"29","author":"Dobin","year":"2013","journal-title":"Bioinformatics"},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"1859","DOI":"10.1093\/bioinformatics\/bti310","article-title":"GMAP: A genomic mapping and alignment program for mRNA and EST sequences","volume":"21","author":"Wu","year":"2005","journal-title":"Bioinformatics"},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"4570","DOI":"10.1093\/nar\/gkq211","article-title":"Detection of splice junctions from paired-end RNA-seq data by SpliceMap","volume":"38","author":"Au","year":"2010","journal-title":"Nucleic Acids Res."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"1837","DOI":"10.1093\/bioinformatics\/btu146","article-title":"Lacking alignments? The next-generation sequencing mapper segemehl revisited","volume":"30","author":"Otto","year":"2014","journal-title":"Bioinformatics"},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"1185","DOI":"10.1038\/nmeth.2221","article-title":"The GEM mapper: Fast, accurate and versatile alignment by filtration","volume":"9","author":"Sammeth","year":"2012","journal-title":"Nat. Methods"},{"key":"ref_34","doi-asserted-by":"crossref","unstructured":"Trincado, J.L., Entizne, J.C., Hysenaj, G., Singh, B., Skalic, M., Elliott, D.J., and Eyras, E. (2018). SUPPA2: Fast, accurate, and uncertainty-aware differential splicing analysis across multiple conditions. Genome Biol., 19.","DOI":"10.1186\/s13059-018-1417-1"},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"187","DOI":"10.1016\/j.molcel.2018.08.018","article-title":"Efficient and Accurate Quantitative Profiling of Alternative Splicing Patterns of Any Complexity on a Laptop","volume":"72","author":"Weatheritt","year":"2018","journal-title":"Mol. Cell"},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"316","DOI":"10.1111\/dgd.12608","article-title":"Nanopore sequencing: Review of potential applications in functional genomics","volume":"61","author":"Kono","year":"2019","journal-title":"Dev. Growth Differ."},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"278","DOI":"10.1016\/j.gpb.2015.08.002","article-title":"PacBio Sequencing and Its Applications","volume":"13","author":"Rhoads","year":"2015","journal-title":"Genom. Proteom. Bioinform."},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"1600","DOI":"10.1080\/15476286.2021.1872961","article-title":"Target-capture full-length double-strand cDNA sequencing for alternative splicing analysis","volume":"18","author":"Ura","year":"2021","journal-title":"RNA Biol."},{"key":"ref_39","doi-asserted-by":"crossref","unstructured":"Ura, H., Togi, S., and Niida, Y. (2023). Target-capture full-length double-stranded cDNA long-read sequencing through Nanopore revealed novel intron retention in patient with tuberous sclerosis complex. Front. Genet., 14.","DOI":"10.3389\/fgene.2023.1256064"},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"2078","DOI":"10.1093\/bioinformatics\/btp352","article-title":"The Sequence Alignment\/Map format and SAMtools","volume":"25","author":"Li","year":"2009","journal-title":"Bioinformatics"},{"key":"ref_41","doi-asserted-by":"crossref","unstructured":"Shen, W., Le, S., Li, Y., and Hu, F. (2016). SeqKit: A Cross-Platform and Ultrafast Toolkit for FASTA\/Q File Manipulation. PLoS ONE, 11.","DOI":"10.1371\/journal.pone.0163962"},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"2184","DOI":"10.1093\/bioinformatics\/bts356","article-title":"RSeQC: Quality control of RNA-seq experiments","volume":"28","author":"Wang","year":"2012","journal-title":"Bioinformatics"},{"key":"ref_43","doi-asserted-by":"crossref","first-page":"178","DOI":"10.1093\/bib\/bbs017","article-title":"Integrative Genomics Viewer (IGV): High-performance genomics data visualization and exploration","volume":"14","author":"Thorvaldsdottir","year":"2013","journal-title":"Brief. Bioinform."}],"container-title":["International Journal of Molecular Sciences"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1422-0067\/26\/7\/3220\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,9]],"date-time":"2025-10-09T17:06:09Z","timestamp":1760029569000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1422-0067\/26\/7\/3220"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2025,3,30]]},"references-count":43,"journal-issue":{"issue":"7","published-online":{"date-parts":[[2025,4]]}},"alternative-id":["ijms26073220"],"URL":"https:\/\/doi.org\/10.3390\/ijms26073220","relation":{},"ISSN":["1422-0067"],"issn-type":[{"value":"1422-0067","type":"electronic"}],"subject":[],"published":{"date-parts":[[2025,3,30]]}}}