Large-scale single-cell long-read genomics enables high-resolution microbiome profiling

Large-scale single-cell long-read genomics enables high-resolution microbiome profiling | 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 Biological Sciences - Article Large-scale single-cell long-read genomics enables high-resolution microbiome profiling Yifan Liu, Mengzhen Li, Shiyan Li, Hengxin Liu, Xuanpei Zhai, and 11 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7858644/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Microbial communities are extraordinarily diverse and play crucial roles in health and disease, yet current methods lack the resolution and scalability needed to dissect their genomic and ecological complexity at the single-cell level. Here, we present CAP-seq, a high-throughput single-microbe genomics platform that combines hydrogel-based semi-permeable encapsulation with moderate microfluidics to recover thousands of single-amplified genomes (SAGs) with long reads and high completeness at moderate sequencing depth. We benchmarked CAP-seq using defined microbial communities, demonstrating strain-level resolution, accurate detection of rare taxa, and genome recovery exceeding 50% at ~10× coverage. Applying CAP-seq to pediatric Clostridioides difficile infection microbiomes, we generated a high-resolution single-cell atlas comprising tens of thousands of SAGs across hundreds of species. Host-resolved profiling of the cryptic plasmid pBI143 revealed previously hidden low-abundance host associations, four new plasmid versions, and their coexistence within individuals, indicating complex plasmid evolution in situ. Longitudinal analysis during fecal microbiota transplantation and vancomycin treatment uncovered dynamic remodeling of microbial hosts, antimicrobial resistance genes, and plasmid-microbial interactions at single-cell resolution. CAP-seq enables scalable, high-performance single-cell genomics and provides a practical, widely accessible platform for microbiome analysis, paving the way for large-scale exploration of microbial dark matter, host–microbe interactions, and plasmid-microbe dynamics across diverse ecosystems. Biological sciences/Biotechnology/Genomics Biological sciences/Microbiology/Microbial communities/Microbiome single-cell genomics microbiome metagenomics plasmid Clostridioides difficile infection Full Text Additional Declarations Yes there is potential Competing Interest. Liu, Y. Du, J. Li, and R. Zhang are co-inventors on a patent application filed by ShanghaiTech University based on the CAP-seq workflow described in this article. Y. Liu and W. Wei are co-founders of a startup company aimed at commercializing CAP-seq. Supplementary Files SupTable1.xlsx Supplementary Table 1 SupMovie1.mp4 Supplementary Movie 1 Cite Share Download PDF Status: Posted 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-7858644","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Biological Sciences - Article","associatedPublications":[],"authors":[{"id":529462137,"identity":"4d3dad3c-9c46-4e3c-923a-772e21df86c6","order_by":0,"name":"Yifan 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Here, we present CAP-seq, a high-throughput single-microbe genomics platform that combines hydrogel-based semi-permeable encapsulation with moderate microfluidics to recover thousands of single-amplified genomes (SAGs) with long reads and high completeness at moderate sequencing depth. We benchmarked CAP-seq using defined microbial communities, demonstrating strain-level resolution, accurate detection of rare taxa, and genome recovery exceeding 50% at ~10× coverage. Applying CAP-seq to pediatric Clostridioides difficile infection microbiomes, we generated a high-resolution single-cell atlas comprising tens of thousands of SAGs across hundreds of species. Host-resolved profiling of the cryptic plasmid pBI143 revealed previously hidden low-abundance host associations, four new plasmid versions, and their coexistence within individuals, indicating complex plasmid evolution in situ. 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