Demultiplexing and barcode-specific adaptive sampling for nanopore direct RNA sequencing

Demultiplexing and barcode-specific adaptive sampling for nanopore direct RNA sequencing | 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 Demultiplexing and barcode-specific adaptive sampling for nanopore direct RNA sequencing Max von Kleist, Wiep van der Toorn, Wang Liu-Wei, Patrick Bohn, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4783223/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 21 Apr, 2025 Read the published version in Nature Communications → Version 1 posted You are reading this latest preprint version Abstract Nanopore direct RNA sequencing (dRNA-seq) enables unique insights into (epi-)transcriptomics. However, applications are currently limited by the lack of accurate and cost-effective sample multiplexing. We introduce WarpDemuX, an ultra-fast and highly accurate adapter-barcoding and demultiplexing approach. WarpDemuX enhances speed and accuracy by fast processing of the raw nanopore signal, use of a light-weight machine-learning algorithm and design of optimized barcode sets. We demonstrate its utility by performing a rapid phenotypic profiling of different SARS-CoV-2 viruses, crucial for pandemic prevention and response, through multiplexed sequencing of longitudinal samples on a single flowcell. This identifies systematic differences in transcript abundance and poly(A) tail lengths during infection. Additionally, integrating WarpDemuX into sequencing control software enables real-time enrichment of target molecules through barcode-specific adaptive sampling, which we demonstrate by enriching low abundance viral RNA. In summary, WarpDemuX is a broadly applicable, high-performance, and economical multiplexing solution for nanopore dRNA-seq, facilitating advanced (epi-)transcriptomic research. Biological sciences/Computational biology and bioinformatics/Software Biological sciences/Molecular biology/Transcriptomics Biological sciences/Microbiology/Virology/SARS-CoV-2 Full Text Additional Declarations Yes there is potential Competing Interest. An international priority patent application was filed jointly by RKI, HZI and FU Berlin on April 26, 2024, at the European Patent Office (EPO) under number PCT/EP2024/061629 describing the method for barcode design and demultiplexing. Supplementary Files WarpDemux240722linenumbersSI.pdf Cite Share Download PDF Status: Published Journal Publication published 21 Apr, 2025 Read the published version in Nature Communications → 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-4783223","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":334994403,"identity":"0bc88027-7a27-4634-b41d-ec81e6e00a20","order_by":0,"name":"Max von 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