{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,25]],"date-time":"2026-09-25T09:04:06Z","timestamp":1790327046025,"version":"4.1.0"},"reference-count":38,"publisher":"PeerJ","license":[{"start":{"date-parts":[[2025,10,30]],"date-time":"2025-10-30T00:00:00Z","timestamp":1761782400000},"content-version":"unspecified","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/100000006","name":"The Office of Naval Research","doi-asserted-by":"crossref","award":["N00014-22-1-2719"],"award-info":[{"award-number":["N00014-22-1-2719"]}],"id":[{"id":"10.13039\/100000006","id-type":"DOI","asserted-by":"crossref"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"abstract":"<jats:p>\n                    There are many decisions to be made when sampling for environmental DNA (eDNA) analysis, whether using a targeted, single-species assay or community-based metabarcoding. Of the entire workflow from sampling water to bioinformatic analyses, the first steps in the process of collecting water, filtering it, and preserving the filter membranes represent major decision points upon which the success of downstream processes depend. Though many previous studies have compared water volume filtered, filter pore size, and preservation and extraction methods, the conclusions are often that they produce different results, but it is unclear which is the optimal approach for a given purpose. Here, rather than provide yet another methods comparison paper, we provide a framework for how to make informed decisions from a methods comparison and, importantly, how to combine data collected\n                    <jats:italic>via<\/jats:italic>\n                    different methodological choices. We investigate (1) the volume of water filtered and the filter pore size and (2) the preservation method and extraction method of samples with a specific lens on how these choices impact the detection of a single targeted species (Atlantic bottlenose dolphin,\n                    <jats:italic>Tursiops truncatus<\/jats:italic>\n                    ,\n                    <jats:italic>via<\/jats:italic>\n                    quantitative PCR (qPCR)), although in principle these findings apply to single-species assays more generally. We find that larger pore size filters (5 \u00b5m\n                    <jats:italic>vs.<\/jats:italic>\n                    1 \u00b5m) and larger volumes of water (3 L\n                    <jats:italic>vs.<\/jats:italic>\n                    1 L) maximize the ratio of amplifiable target DNA to total DNA without compromising the absolute detection of target. We also find that maximizing total DNA yield during extraction (phenol chloroform\n                    <jats:italic>vs.<\/jats:italic>\n                    two commercial kits) does not always increase target detection likely due to the concentration of inhibitors and co-extraction of off-target DNA. We also comment on variation including technical and biological variability between replicates, finding that by homogenizing source water before filtering removes much of the biological variation. Finally, we present a statistical model that allows for inclusion of data from samples collected and processed in different ways, enabling researchers to change protocols or include data from other field sampling efforts, thereby opening up more possibilities to extend datasets and analyses.\n                  <\/jats:p>","DOI":"10.7717\/peerj.20127","type":"journal-article","created":{"date-parts":[[2025,10,30]],"date-time":"2025-10-30T08:19:15Z","timestamp":1761812355000},"page":"e20127","source":"Crossref","is-referenced-by-count":10,"title":["Optimizing target-to-total DNA ratio in eDNA studies: effects of sampling, preservation, and extraction methods on single-species detection"],"prefix":"10.7717","volume":"13","author":[{"given":"Elizabeth","family":"Andruszkiewicz Allan","sequence":"first","affiliation":[{"name":"School of Marine and Environmental Affairs, University of Washington, Seattle, WA,  United States of America"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Megan R.","family":"Shaffer","sequence":"additional","affiliation":[{"name":"School of Marine and Environmental Affairs, University of Washington, Seattle, WA,  United States of America"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ryan P.","family":"Kelly","sequence":"additional","affiliation":[{"name":"School of Marine and Environmental Affairs, University of Washington, Seattle, WA,  United States of America"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Kim","family":"Parsons","sequence":"additional","affiliation":[{"name":"Conservation Biology Division, Northwest Fisheries Science Center, National Marine Fisheries Service, National Oceanic and Atmospheric Administration, Seattle, WA, United States of America"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"4443","published-online":{"date-parts":[[2025,10,30]]},"reference":[{"key":"10.7717\/peerj.20127\/ref-1","doi-asserted-by":"publisher","first-page":"493","DOI":"10.1002\/edn3.74","article-title":"Maximizing fish detection with eDNA metabarcoding","volume":"2","author":"Bessey","year":"2020","journal-title":"Environmental DNA"},{"key":"10.7717\/peerj.20127\/ref-2","doi-asserted-by":"publisher","first-page":"e570","DOI":"10.1002\/edn3.570","article-title":"Enhancing environmental DNA metabarcoding from marine ecosystems: impact of filter type, storage method, and storage time on the assessment of fish alpha and beta diversity","volume":"6","author":"Bizzozzero","year":"2024","journal-title":"Environmental DNA"},{"key":"10.7717\/peerj.20127\/ref-3","doi-asserted-by":"publisher","first-page":"e560","DOI":"10.1002\/edn3.560","article-title":"A comparison of eDNA sampling methods in an estuarine environment on presence of longfin smelt (Spirinchus thaleichthys) and fish community composition","volume":"6","author":"Bowen","year":"2024","journal-title":"Environmental DNA"},{"key":"10.7717\/peerj.20127\/ref-4","doi-asserted-by":"publisher","first-page":"4","DOI":"10.1029\/2024JC021643","article-title":"Marine eDNA production and loss mechanisms","volume":"130","author":"Brasseale","year":"2025","journal-title":"Journal of Geophysical Research: Oceans"},{"key":"10.7717\/peerj.20127\/ref-5","doi-asserted-by":"publisher","first-page":"152","DOI":"10.1002\/edn3.52","article-title":"Effects of filtration methods and water volume on the quantification of brown trout (Salmo trutta) and Arctic char (Salvelinus alpinus) eDNA concentrations via droplet digital PCR","volume":"2","author":"Capo","year":"2020","journal-title":"Environmental DNA"},{"key":"10.7717\/peerj.20127\/ref-6","doi-asserted-by":"publisher","first-page":"214","DOI":"10.1016\/j.mito.2020.06.004","article-title":"Thinking outside the nucleus: mitochondrial DNA copy number in health and disease","volume":"53","author":"Castellani","year":"2020","journal-title":"Mitochondrion"},{"key":"10.7717\/peerj.20127\/ref-7","doi-asserted-by":"publisher","first-page":"619","DOI":"10.1002\/edn3.158","article-title":"Detecting fish assemblages with environmental DNA: does protocol matter? Testing eDNA metabarcoding method robustness","volume":"3","author":"Coutant","year":"2021","journal-title":"Environmental DNA"},{"key":"10.7717\/peerj.20127\/ref-8","doi-asserted-by":"publisher","first-page":"e28963","DOI":"10.3897\/mbmg.2.28963","article-title":"Optimising the detection of marine taxonomic richness using environmental DNA metabarcoding: the effects of filter material, pore size and extraction method","volume":"2","author":"Deiner","year":"2018","journal-title":"Metabarcoding and Metagenomics"},{"key":"10.7717\/peerj.20127\/ref-9","doi-asserted-by":"publisher","first-page":"53","DOI":"10.1016\/j.biocon.2014.11.018","article-title":"Choice of capture and extraction methods affect detection of freshwater biodiversity from environmental DNA","volume":"183","author":"Deiner","year":"2015","journal-title":"Biological 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review of the macro-organismal biomonitoring revolution","volume":"873","author":"Takahashi","year":"2023","journal-title":"Science of the Total Environment"},{"key":"10.7717\/peerj.20127\/ref-34","doi-asserted-by":"publisher","first-page":"1136","DOI":"10.1111\/2041-210X.13212","article-title":"A self-preserving, partially biodegradable eDNA filter","volume":"10","author":"Thomas","year":"2019","journal-title":"Methods in Ecology and Evolution"},{"key":"10.7717\/peerj.20127\/ref-35","doi-asserted-by":"publisher","first-page":"99","DOI":"10.1002\/edn3.21","article-title":"The detection of aquatic macroorganisms using environmental DNA analysis: a review of methods for collection, extraction, and detection","volume":"1","author":"Tsuji","year":"2019","journal-title":"Environmental DNA"},{"key":"10.7717\/peerj.20127\/ref-36","doi-asserted-by":"publisher","first-page":"e114329","DOI":"10.1371\/journal.pone.0114329","article-title":"Improved methods for capture, extraction, and quantitative assay of environmental DNA from Asian Bigheaded Carp (Hypophthalmichthys spp.)","volume":"9","author":"Turner","year":"2014","journal-title":"PLOS ONE"},{"key":"10.7717\/peerj.20127\/ref-37","doi-asserted-by":"publisher","first-page":"789","DOI":"10.1007\/s12686-015-0483-x","article-title":"Long duration, room temperature preservation of filtered eDNA samples","volume":"7","author":"Wegleitner","year":"2015","journal-title":"Conservation Genetics Resources"},{"issue":"3","key":"10.7717\/peerj.20127\/ref-38","first-page":"1207","article-title":"High copy number of mitochondrial DNA (mtDNA) predicts good prognosis in glioma patients","volume":"5","author":"Zhang","year":"2015","journal-title":"American Journal of Cancer 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