Single-cell multi-omics resolved analysis of mitochondrial genome-wide mutational burden, constraint, and mosaicism

Single-cell multi-omics resolved analysis of mitochondrial genome-wide mutational burden, constraint, and mosaicism | 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 Technical Report Single-cell multi-omics resolved analysis of mitochondrial genome-wide mutational burden, constraint, and mosaicism Leif Ludwig, Yu-Hsin Hsieh, Pauline Kautz, Lena Nitsch, Ambre Giguelay, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6539733/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 16 Mar, 2026 Read the published version in Nature Communications → Version 1 posted You are reading this latest preprint version Abstract Mitochondria contain their own circular, multi-copy genome, encoding essential components of the mitochondrial respiratory chain vital for cellular metabolism. Mitochondrial DNA (mtDNA) mutations occur more frequently than nuclear mutations and are associated with various diseases. While single-cell sequencing has allowed analysis of mtDNA variant heteroplasmy, a holistic view of mtDNA mutational landscapes in individual cells has remained limited, prohibiting the investigation of fundamental questions in mitochondrial genetics. Here, we leverage mitochondrial single-cell ATAC-seq (mtscATAC-seq) and mtDNA-hypermutated POLGD274A knock-in HEK293 cell lines to introduce two metrics—single-cell mtDNA mutations per million base pairs (scmtMPM) and heteroplasmy-weighted mitochondrial local constraint scores (scwMSS)—to capture cellular mutational loads and somatic mosaicism. We demonstrate that individual POLGD274A cells are characterized by complex mutational landscapes, with pathogenic mutations and truncating variants only present at subthreshold levels, indicative of their negative selection. In human healthy donors and mitochondriopathy patients, we identify constrained mutations in complex I, highlighting previously unrecognized mtDNA mutational landscape heterogeneity present on the single-cell level. Overall, scmtMPM and scwMSS provide a novel framework to investigate fundamental properties of mitochondrial genetics, disease, and somatic mosaicism in human tissues. Biological sciences/Genetics/Genomics Biological sciences/Genetics/Sequencing/DNA sequencing Biological sciences/Computational biology and bioinformatics/Genome informatics Biological sciences/Systems biology/Systems analysis Health sciences/Medical research/Genetics research Full Text Additional Declarations Yes there is potential Competing Interest. The Broad Institute has filed for patents relating to the use of technologies described in this paper, where CAL and LSL are named inventors (US patent applications 17/251,451 and 17/928,696). CAL is a consultant to Cartography Biosciences. Cite Share Download PDF Status: Published Journal Publication published 16 Mar, 2026 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-6539733","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Technical Report","associatedPublications":[],"authors":[{"id":454484591,"identity":"ca3bce0f-333e-425a-bf03-eba06e7d7660","order_by":0,"name":"Leif Ludwig","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABDklEQVRIie2QMUvEMBiG31BolqBrQIp/IdBBRPG3NAjncoIgHOJyKUJdlK53/6KTuF2kcF3EUQo32CzOB05uxvQ8XHI6CuYZPkL4Ht68AQKBP8gWiFrizM4IpEMMCIDozA7lUWKQfGLXPpVIrBT8oID0ij3ztYJNCr3KFUSdVA19vmxHR9ijt50290hKn8IenJJWNRsthk/H2L9phJaPSKeemJjL684q8q5mg8VpocdVO4CWBWSlPcqucSlOObcKxMtrr8x8CidfCp1HTmnjVYq3i8xVJk7SWc2inb6LexhPJ56UbdoYtbw4SKZlY96G7sfmxLwXh0npqe/I3GTi+x3fsL+Gdr/ZCgQCgX/IBy2fYlKMil9zAAAAAElFTkSuQmCC","orcid":"https://orcid.org/0000-0002-2916-2164","institution":"Berlin Institute of Health / Berlin Institute for Medical Systems Biology","correspondingAuthor":true,"prefix":"","firstName":"Leif","middleName":"","lastName":"Ludwig","suffix":""},{"id":454484592,"identity":"0f5b1d58-7cae-4d56-84bb-7c36f99bfe12","order_by":1,"name":"Yu-Hsin Hsieh","email":"","orcid":"","institution":"Berlin Institute of Health / Berlin Institute of Medical Systems Biology","correspondingAuthor":false,"prefix":"","firstName":"Yu-Hsin","middleName":"","lastName":"Hsieh","suffix":""},{"id":454484593,"identity":"b04f807e-068e-4868-966e-4afb4dd8e33c","order_by":2,"name":"Pauline Kautz","email":"","orcid":"","institution":"Berlin Institute of Health at Charité – Universitätsmedizin Berlin","correspondingAuthor":false,"prefix":"","firstName":"Pauline","middleName":"","lastName":"Kautz","suffix":""},{"id":454484594,"identity":"b00a34fe-5a01-49cb-93b0-a5bb6bcb54c8","order_by":3,"name":"Lena Nitsch","email":"","orcid":"","institution":"Berlin Institute of Health / Berlin Institute of Medical Systems Biology","correspondingAuthor":false,"prefix":"","firstName":"Lena","middleName":"","lastName":"Nitsch","suffix":""},{"id":454484595,"identity":"028531fe-e9b9-49f7-9a1d-22bf888092d2","order_by":4,"name":"Ambre Giguelay","email":"","orcid":"https://orcid.org/0000-0003-0417-1109","institution":"Berlin Institute of Health at Charité - Universitätsmedizin Berlin","correspondingAuthor":false,"prefix":"","firstName":"Ambre","middleName":"","lastName":"Giguelay","suffix":""},{"id":454484596,"identity":"fa0b27dd-cee8-4946-a0ad-76b4191d97b4","order_by":5,"name":"Janet Liebold","email":"","orcid":"","institution":"Berlin Institute of Health at Charité – Universitätsmedizin Berlin","correspondingAuthor":false,"prefix":"","firstName":"Janet","middleName":"","lastName":"Liebold","suffix":""},{"id":454484597,"identity":"23ba9308-8484-41f8-a9f5-4ce2aea85794","order_by":6,"name":"Gábor Zsurka","email":"","orcid":"https://orcid.org/0000-0002-6379-849X","institution":"University of Bonn","correspondingAuthor":false,"prefix":"","firstName":"Gábor","middleName":"","lastName":"Zsurka","suffix":""},{"id":454484598,"identity":"6d1d7286-9c1c-4840-9197-ad58190ac699","order_by":7,"name":"Genevieve Trombly","email":"","orcid":"","institution":"University Bonn Medical Center","correspondingAuthor":false,"prefix":"","firstName":"Genevieve","middleName":"","lastName":"Trombly","suffix":""},{"id":454484599,"identity":"170b2827-fbf3-48bd-bc23-15069422eebb","order_by":8,"name":"Markus Schuelke","email":"","orcid":"https://orcid.org/0000-0003-2824-3891","institution":"Charité - Universitätsmedizin Berlin, corporate member of Freie Universität Berlin and Humboldt-Universität zu Berlin; 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