Quantitative evaluation of microbiome sequencing resolution under varying experimental conditions using defined mock communities

Quantitative evaluation of microbiome sequencing resolution under varying experimental conditions using defined mock communities | 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 Quantitative evaluation of microbiome sequencing resolution under varying experimental conditions using defined mock communities Songhee Lee, Hyeonah Lee, Jung Wook Kim, Hyeon-Jin Kim, Kwang Jun Lee This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8390039/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 15 You are reading this latest preprint version Abstract Background : Objective evaluation of sequencing resolution is crucial for comparing technologies and ensuring reproducibility in microbiome analysis. Specifically, a systematic approach is necessary to quantitatively assess the effect of various platforms and experimental conditions on species-level resolution. Therefore, this study quantitatively evaluated multiple strategies, including 16S V3–V4 (16P), full-length 16S rRNA gene (16F), and whole metagenome shotgun sequencing (WMS), using a commercial DNA-based mock community (MC) and a domestically developed whole-cell MC (Korea MC [KMC]). The WMS strategy included 12 combinations of input DNA concentrations and sequencing output levels. A total of 64 WMS libraries were constructed for KMC samples, and 112 sequencing datasets were analysed. Taxonomic resolution was assessed using an adjusted F1-score integrating detection sensitivity and abundance-level reproducibility. Results : Qualitatively examining the detected species against the expected species across platforms, WMS showed a true positive abundance ratio of over 90%, 16F was observed to have an average of 60%, and 16P was observed to have an average of less than 10%. The combination of 10 ng input and 10 gigabases output consistently yielded the highest species-level resolution. However, reduced performance was observed in some MCs under 1 ng or 100 ng DNA input conditions. Detection sensitivity varied by taxon and condition. Specifically, Streptococcus pneumoniae and Cryptococcus neoformans were detected only under high-input or -output conditions, whereas Escherichia coli exhibited optimal accuracy at intermediate inputs. Acinetobacter species demonstrated reduced resolution as input DNA increased. KMC samples showed species- and format-specific variability in DNA extraction efficiency. Conclusions : This study establishes a quantitative framework for assessing species-level resolution across sequencing conditions and taxa using defined MCs. The findings provide practical guidance for selecting sequencing strategies aligned with analytical objectives and resource constraints. Biological sciences/Biological techniques Biological sciences/Computational biology and bioinformatics Biological sciences/Microbiology Mock community Sequencing strategy Metagenomics 16S rRNA gene Taxonomic resolution Full Text Additional Declarations No competing interests reported. Supplementary Files Supplymentarymaterial.pdf Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 23 Jan, 2026 Reviews received at journal 18 Jan, 2026 Reviews received at journal 18 Jan, 2026 Reviews received at journal 14 Jan, 2026 Reviewers agreed at journal 11 Jan, 2026 Reviewers agreed at journal 08 Jan, 2026 Reviewers agreed at journal 08 Jan, 2026 Reviewers agreed at journal 08 Jan, 2026 Reviews received at journal 04 Jan, 2026 Reviewers agreed at journal 29 Dec, 2025 Reviewers invited by journal 26 Dec, 2025 Editor invited by journal 22 Dec, 2025 Editor assigned by journal 18 Dec, 2025 Submission checks completed at journal 18 Dec, 2025 First submitted to journal 17 Dec, 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-8390039","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":566598572,"identity":"2169115d-6a06-4b53-96fa-fee1f1bda184","order_by":0,"name":"Songhee Lee","email":"","orcid":"","institution":"Korea National Institute of Health","correspondingAuthor":false,"prefix":"","firstName":"Songhee","middleName":"","lastName":"Lee","suffix":""},{"id":566598573,"identity":"ff0bfa72-6908-469f-910b-c3843c169a61","order_by":1,"name":"Hyeonah Lee","email":"","orcid":"","institution":"Korea National Institute of 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community, Sequencing strategy, Metagenomics, 16S rRNA gene, Taxonomic resolution","lastPublishedDoi":"10.21203/rs.3.rs-8390039/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8390039/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground\u003c/strong\u003e: Objective evaluation of sequencing resolution is crucial for comparing technologies and ensuring reproducibility in microbiome analysis. 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