Technical considerations for cost-effective Transposon Directed Insertion-Site Sequencing (TraDIS)

Technical considerations for cost-effective Transposon Directed Insertion-Site Sequencing (TraDIS) | 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 Technical considerations for cost-effective Transposon Directed Insertion-Site Sequencing (TraDIS) Yasuhiro Kyono, Madeline Tolwinski, Stephanie A. Flowers This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3784942/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 21 Mar, 2024 Read the published version in Scientific Reports → Version 1 posted 10 You are reading this latest preprint version Abstract Transposon Directed Insertion-Site Sequencing (TraDIS) is a high-throughput assay that defines essential bacterial genes across diverse growth conditions. However, the variability between laboratory environments often requires laborious, time-consuming modifications to its protocol. In this technical study, we aimed to refine the protocol by identifying key parameters that can impact the complexity of mutant libraries. Firstly, we discovered that adjusting electroporation parameters including transposome concentration, transposome assembly conditions, and cell densities can significantly improve the recovery of viable mutants for different Escherichia coli strains. Secondly, we found that post-electroporation conditions, such as recovery time and the use of different mediums for selecting mutants may also impact the complexity of viable mutants in the library. Finally, we developed a simplified sequencing library preparation workflow based on a Nextera-TruSeq hybrid design without compromising the ability to detect transposon-DNA junctions. The technical improvements presented in our study aim to streamline TraDIS protocols, making this powerful technique more accessible for a wider scientific audience. Biological sciences/Genetics Biological sciences/Microbiology Biological sciences/Molecular biology Transposon insertion sequencing Escherichia coli Electroporation Illumina Library Preparation Full Text Additional Declarations No competing interests reported. Supplementary Files SupplementalTable.xlsx Cite Share Download PDF Status: Published Journal Publication published 21 Mar, 2024 Read the published version in Scientific Reports → Version 1 posted Editorial decision: Revision requested 31 Jan, 2024 Reviews received at journal 21 Jan, 2024 Reviews received at journal 05 Jan, 2024 Reviewers agreed at journal 02 Jan, 2024 Reviewers agreed at journal 30 Dec, 2023 Reviewers invited by journal 28 Dec, 2023 Editor assigned by journal 22 Dec, 2023 Editor invited by journal 22 Dec, 2023 Submission checks completed at journal 22 Dec, 2023 First submitted to journal 21 Dec, 2023 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. 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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-3784942","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":262640643,"identity":"583e689c-7147-4d70-ad3a-6f59243f44f7","order_by":0,"name":"Yasuhiro Kyono","email":"","orcid":"","institution":"University of Illinois at Chicago","correspondingAuthor":false,"prefix":"","firstName":"Yasuhiro","middleName":"","lastName":"Kyono","suffix":""},{"id":262640646,"identity":"66fde13f-0129-457f-a8bf-f45f0a496899","order_by":1,"name":"Madeline Tolwinski","email":"","orcid":"","institution":"University of Illinois at Chicago","correspondingAuthor":false,"prefix":"","firstName":"Madeline","middleName":"","lastName":"Tolwinski","suffix":""},{"id":262640647,"identity":"728b6d63-2903-4e00-b886-7038ad24b7bf","order_by":2,"name":"Stephanie A. 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