Variation of and associations with the depth and evenness of sequencing coverage in archived plastid genomes

Variation of and associations with the depth and evenness of sequencing coverage in archived plastid genomes | 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 Variation of and associations with the depth and evenness of sequencing coverage in archived plastid genomes Nils Jenke, Gregory M. Smith, Buddha Thapa Magar, Michael Gruenstaeudl This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5784537/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 19 Jul, 2025 Read the published version in Scientific Reports → Version 1 posted 6 You are reading this latest preprint version Abstract Depth and evenness of sequencing coverage are considered potential indicators of genome assembly quality. In plastid genomics, where new data generation has outpaced the development of assembly quality indicators, these coverage metrics could offer insights into the quality of plastomes of different sizes, structures, or taxonomic origins. However, the variation of sequencing depth and evenness among archived plastid genomes, their variability between genome partitions, and any association with methodological factors have yet to be evaluated. This study explores the variation of sequencing depth and evenness across a sample of publicly accessible plastid genomes in relation to their genome structure, assembly quality, and methodological provenance using uni- and multivariate statistical analyses. We also evaluate whether sequencing evenness in plastid genomes is biased by phylogenetic signal and assembly software choice, and whether more uniformly distributed input sequence data improves plastome assembly quality. Our results indicate significant differences in sequencing depth across the four structural partitions and between the coding and non-coding regions of plastid genomes, a significant correlation between sequencing evenness and the number of ambiguous nucleotides, and a significant difference in sequencing evenness between sequencing platforms. However, we also find that different covariates representing additional, lesser explored factors often show a similar, if not greater, explanatory power for the coverage variation. No indications of phylogenetic or software choice bias on sequencing evenness and only weak indications of phylogenetic bias among the assembly quality metrics are detected, suggesting that our study results represent genuine patterns. We also find that normalizing the distribution of the input sequence data before plastome assembly may improve assembly accuracy. Taken together, these findings highlight that many public plastid genomes derive from sequence data with highly variable depth and evenness, and that this variation is influenced, at least partially, by genome structure as well as methodological factors. Biological sciences/Genetics/Genomics/Comparative genomics Biological sciences/Computational biology and bioinformatics/Data mining Full Text Additional Declarations No competing interests reported. Supplementary Files JenkeEtAlManuscriptrevisedSupplMaterials.pdf Cite Share Download PDF Status: Published Journal Publication published 19 Jul, 2025 Read the published version in Scientific Reports → Version 1 posted Editorial decision: Accepted 10 Jul, 2025 Reviews received at journal 02 Jul, 2025 Reviewers agreed at journal 20 Jun, 2025 Reviewers invited by journal 19 Jun, 2025 Submission checks completed at journal 17 Jun, 2025 First submitted to journal 09 Jun, 2025 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. 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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-5784537","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":483652237,"identity":"edc5de5c-4087-489a-9ca6-4d3e2ce2b31f","order_by":0,"name":"Nils Jenke","email":"","orcid":"","institution":"Freie Universität Berlin","correspondingAuthor":false,"prefix":"","firstName":"Nils","middleName":"","lastName":"Jenke","suffix":""},{"id":483652238,"identity":"d262c8ab-a7ad-4373-ac86-fd31a41d653e","order_by":1,"name":"Gregory M. 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