Disentangled assembly graphs reveal hidden eukaryotic diversity in metagenomic data

Disentangled assembly graphs reveal hidden eukaryotic diversity in metagenomic data | Authorea try { document.documentElement.classList.add('js'); } catch (e) { } var _gaq = _gaq || []; _gaq.push(['_setAccount', 'G-8VDV14Y67G']); _gaq.push(['_trackPageview']); (function() { var ga = document.createElement('script'); ga.type = 'text/javascript'; ga.async = true; ga.src = ('https:' == document.location.protocol ? 'https://ssl' : 'http://www') + '.google-analytics.com/ga.js'; var s = document.getElementsByTagName('script')[0]; s.parentNode.insertBefore(ga, s); })(); Skip to main content Preprints Collections Wiley Open Research IET Open Research Ecological Society of Japan All Collections About About Authorea FAQs Contact Us Quick Search anywhere Search for preprint articles, keywords, etc. Search Search ADVANCED SEARCH SCROLL Molecular Ecology Resources This is a preprint and has not been peer reviewed. Data may be preliminary. 3 September 2025 V1 Latest version Share on Disentangled assembly graphs reveal hidden eukaryotic diversity in metagenomic data Authors : Manon Geerts 0000-0002-5709-3004 [email protected] , Manuel Curto 0000-0002-1630-4653 , Andrew Alverson 0000-0003-1241-2654 , Jeffery Stone , and Hugo Gante Authors Info & Affiliations https://doi.org/10.22541/au.175692108.88944515/v1 Published Molecular Ecology Resources Version of record Peer review timeline 255 views 208 downloads Contents Abstract Supplementary Material Information & Authors Metrics & Citations View Options References Figures Tables Media Share Abstract Assembly graphs contain valuable yet frequently overlooked information that can enhance assembly completeness and accuracy by revealing contig connectivity. Here, we demonstrate how leveraging these information-rich structures enables the discovery of hidden diatom diversity in environmental DNA shotgun datasets. While GetOrganelle has previously been used for organellar genome assembly from isolated tissues, we present its first application to water metagenomic data. We tested the efficiency of this tool on three eDNA samples with varying diatom abundances, finding that GetOrganelle alone often results in fragmented scaffolds due to the complexity of mixed-species samples. By implementing additional manual disentanglement of assembly graphs, we successfully recovered fully complete plastome structures. From high-abundance bloom samples, we recovered complete plastomes of Stephanodiscus hantzschii (129,551 bp and 129,553 bp) with 99.9% pairwise identity, despite originating from distinct geographical locations (USA and Czech Republic). From a lower-abundance non-bloom sample, we reconstructed a potentially novel Cyclotella species plastome (133,867 bp) showing only 92.4% similarity to its closest available reference Cyclotella atomus. Our assembly quality assessment confirmed effective manual disentanglement of target sequences from complex assembly graphs, even when abundance was low. By integrating sequence similarity, gene order conservation, and phylogenetic analysis, we achieved robust species-level resolution and resolved taxonomic uncertainties previously identified. Our findings demonstrate that mining metagenomic data with GetOrganelle reveals previously hidden diversity and provides higher taxonomic resolution than traditional methods. This approach proves especially valuable for diatoms and other microeukaryotes, where reference organellar genomes remain severely underrepresented in existing databases. Supplementary Material File (organellarmetagnomics_maindoc.docx) Download 4.68 MB File (organellarmetagnomics_suppltables.xlsx) Download 64.67 KB Information & Authors Information Version history V1 Version 1 03 September 2025 Peer review timeline Published Molecular Ecology Resources Version of Record 20 Mar 2026 Published Copyright This work is licensed under a Non Exclusive No Reuse License. Collection Molecular Ecology Resources Keywords assembly diatoms environmental dna metagenomics microeukaryotes organellar genomes Authors Affiliations Manon Geerts 0000-0002-5709-3004 [email protected] KU Leuven View all articles by this author Manuel Curto 0000-0002-1630-4653 CIBIO – Research Center in Biodiversity and Genetic Resources, InBIO Laboratório Associado, Campus de Vairão, 4485-661 Vairão View all articles by this author Andrew Alverson 0000-0003-1241-2654 University of Arkansas View all articles by this author Jeffery Stone Indiana State University View all articles by this author Hugo Gante KU Leuven View all articles by this author Metrics & Citations Metrics Article Usage 255 views 208 downloads .FvxKWukQNSOunydq8rnd { width: 100px; } Citations Download citation Manon Geerts, Manuel Curto, Andrew Alverson, et al. Disentangled assembly graphs reveal hidden eukaryotic diversity in metagenomic data. Authorea . 03 September 2025. DOI: https://doi.org/10.22541/au.175692108.88944515/v1 If you have the appropriate software installed, you can download article citation data to the citation manager of your choice. Simply select your manager software from the list below and click Download. For more information or tips please see 'Downloading to a citation manager' in the Help menu . Format Please select one from the list RIS (ProCite, Reference Manager) EndNote BibTex Medlars RefWorks Direct import Tips for downloading citations document.getElementById('citMgrHelpLink').addEventListener('click', function() { popupHelp(this.href); return false; }); $(".js__slcInclude").on("change", function(e){ if ($(this).val() == 'refworks') $('#direct').prop("checked", false); $('#direct').prop("disabled", ($(this).val() == 'refworks')); }); View Options View options PDF View PDF Figures Tables Media Share Share Share article link Copy Link Copied! Copying failed. 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