Benchmarking RNA-seq with the Quartet Reference Materials to establish Best Practices for Accurate Alternative Splicing Detection

Benchmarking RNA-seq with the Quartet Reference Materials to establish Best Practices for Accurate Alternative Splicing Detection | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Benchmarking RNA-seq with the Quartet Reference Materials to establish Best Practices for Accurate Alternative Splicing Detection Rui Zhang, Duo Wang, Jiaxin Zhao, Qingwang Chen, Yanxi Han, Yaqing Liu, and 7 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7716867/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted You are reading this latest preprint version Abstract Previous limited characterization of RNA-seq accuracy in alternative splicing (AS) due to methodological diversity and lack of reference standards has left unclear how to achieve optimal performance for informed applications—an issue increasingly critical with the rise of long-read sequencing. To address this, we conducted the first large-scale reference-based benchmarking of AS detection accuracy across 42 laboratories and 159 analysis pipelines, leveraging the Quartet reference materials. We show that high-quality RNA-seq enables relatively accurate isoform detection. Best practices for experimental and bioinformatic designs were identified, and achieved mean Pearson correlations of 0.82 for isoform quantification and Matthews correlation coefficients of 0.69 for differential expression analysis, representing improvements of 0.21–0.31 and 0.46–0.61 across laboratories, respectively, compared to the poorest workflows. AS event-level accuracy remained limited, yet the best tools still outperformed the poorest by 0.02–0.25 in event quantification and 0.10–0.25 in differential splicing analysis. Beyond technical variables, low expression levels were the primary constraint on isoform and event detection accuracy, followed by their compositional complexity. Collectively, this study provides practical guidance for maximizing AS profiling accuracy with existing methodologies, contributing to effective RNA-seq application in splicing research. Biological sciences/Biotechnology/Sequencing/RNA sequencing Biological sciences/Biological techniques/Sequencing/RNA sequencing Biological sciences/Genetics/RNA splicing Biological sciences/Genetics/Gene regulation RNA sequencing Quartet Alternative Splicing Best practices Benchmarking Full Text Additional Declarations There is NO Competing Interest. Supplementary Files SupplementaryData1.xlsx Dataset 1 SupplementaryData3.xlsx Dataset 3 SupplementaryData2.xlsx Dataset 2 SupplementaryData4.xlsx Dataset 4 SupplementaryData6.xlsx Dataset 6 SupplementaryData5.xlsx Dataset 5 Supplementaryinformation.pdf Supplementary information Cite Share Download PDF Status: Under Review Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7716867","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":530536200,"identity":"45154ec5-c445-42fc-98eb-8c3935118040","order_by":0,"name":"Rui 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