Development of PCR Blocking Primers Enabling DNA Metabarcoding Analysis of Dietary Composition in Hematophagous Sea Lamprey

Development of PCR Blocking Primers Enabling DNA Metabarcoding Analysis of Dietary Composition in Hematophagous Sea Lamprey | 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 Search Search ADVANCED SEARCH SCROLL Ecology and Evolution This is a preprint and has not been peer reviewed. Data may be preliminary. 3 September 2025 V1 Latest version Share on Development of PCR Blocking Primers Enabling DNA Metabarcoding Analysis of Dietary Composition in Hematophagous Sea Lamprey Authors : Conor O'Kane 0009-0007-6297-4985 [email protected] , Nicholas Johnson , Kim Scribner 0000-0003-3416-2828 , Weiming Li , Jeannette Kanefsky , and John Robinson Authors Info & Affiliations https://doi.org/10.22541/au.175692107.78192940/v1 Published Ecology and Evolution Version of record Peer review timeline 199 views 175 downloads Contents Abstract Supplementary Material Information & Authors Metrics & Citations View Options References Figures Tables Media Share Abstract Since the establishment of the invasive sea lamprey (Petromyzon marinus) in the Great Lakes, extensive management efforts have attempted to reduce negative impacts on native fishes. Despite a significant reduction in sea lamprey population abundance following application of several control methods, uncertainties remain concerning damage caused by sea lamprey predation on Great Lakes fish populations. While conventional dietary assessments are hindered by the hematophagous nature of sea lamprey, DNA metabarcoding offers a promising alternative by identifying prey species DNA from sea lamprey digestive tract samples. DNA metabarcoding has been used for dietary analyses in numerous species, including lampreys; however, initial assessments using polymerase chain reaction (PCR) primers designed to amplify the 12S rRNA gene in vertebrate taxa indicated a high presence of sea lamprey amplification product per sample. To minimize sea lamprey DNA co-amplification, we designed and tested eight blocking primers that suppress the amplification of sea lamprey 12S sequences while allowing amplification of host species DNA. This approach allowed for the use of a single marker to amplify a taxonomically diverse suite of host species, in contrast to previous studies that used multiple taxon-specific primer pairs (e.g. Salmonidae, Cyprinidae, and Catostomidae). Candidate blocking primers evaluated in this study differed in base pair length, end sequence modification, and purification method. Samples with different sea lamprey-to-host DNA ratios were subjected to multiple detection methods including gel electrophoresis, quantitative PCR, and DNA metabarcoding to assess the ability of each blocking primer to selectively suppress amplification of the sea lamprey 12S gene region. All blocking primers tested performed well and demonstrated high effectiveness. Results show that the blocking primers evaluated can facilitate molecular diet analysis in sea lamprey, allowing the amplification of a taxonomically diverse range of host fish species with universal primers. Supplementary Material File (development of pcr blocking primers enabling dna metabarcoding analysis of dietary composition in hematophagous sea lamprey_okane.docx) Download 3.81 MB Information & Authors Information Version history V1 Version 1 03 September 2025 Peer review timeline Published Ecology and Evolution Version of Record 20 Aug 2025 Published Copyright This work is licensed under a Non Exclusive No Reuse License. Collection Ecology and Evolution Keywords freshwater genetics molecular genetics vertebrate Authors Affiliations Conor O'Kane 0009-0007-6297-4985 [email protected] Michigan State University View all articles by this author Nicholas Johnson US Geological Survey Hammond Bay Biological Station View all articles by this author Kim Scribner 0000-0003-3416-2828 Michigan State University View all articles by this author Weiming Li Michigan State University View all articles by this author Jeannette Kanefsky Michigan State University View all articles by this author John Robinson University of Georgia View all articles by this author Metrics & Citations Metrics Article Usage 199 views 175 downloads .FvxKWukQNSOunydq8rnd { width: 100px; } Citations Download citation Conor O'Kane, Nicholas Johnson, Kim Scribner, et al. Development of PCR Blocking Primers Enabling DNA Metabarcoding Analysis of Dietary Composition in Hematophagous Sea Lamprey. Authorea . 03 September 2025. DOI: https://doi.org/10.22541/au.175692107.78192940/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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