{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,29]],"date-time":"2026-09-29T20:19:49Z","timestamp":1790713189628,"version":"4.1.0"},"reference-count":11,"publisher":"Public Library of Science (PLoS)","issue":"1","license":[{"start":{"date-parts":[[2024,1,2]],"date-time":"2024-01-02T00:00:00Z","timestamp":1704153600000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"Environomics CSIRO Future Science Platform","award":["R-10011 and R-14486"],"award-info":[{"award-number":["R-10011 and R-14486"]}]}],"content-domain":{"domain":["www.plosone.org"],"crossmark-restriction":false},"short-container-title":["PLoS ONE"],"abstract":"<jats:p>Formalin fixation of natural history specimens and histopathological material has historically been viewed as an impediment to successful genomic analysis. However, the development of extraction methods specifically tailored to contend with heavily crosslinked archival tissues, re-contextualises millions of previously overlooked specimens as viable molecular assets. Here, we present an easy-to-follow protocol for screening archival wet specimens for molecular viability and subsequent genomic DNA extraction suitable for sequencing. The protocol begins with non-destructive assessment of specimen degradation and preservation media conditions to allow both museum curators and researchers to select specimens most likely to yield an acceptable proportion (20\u201360%) of mappable endogenous DNA during short-read DNA sequencing. The extraction protocol uses hot alkaline lysis in buffer (0.1M NaOH, 1% SDS, pH 13) to simultaneously lyse and de-crosslink the tissue. To maximise DNA recovery, phenol:chloroform extraction is coupled with a small-fragment optimised SPRI bead clean up. Applied to well-preserved archival tissues, the protocol can yield 1\u20132 \u03bcg DNA per 50 mg of tissue with mean fragment sizes typically ranging from 50\u2013150 bp, which is suitable to recover genomic DNA sufficient to reconstruct complete mitochondrial genomes and achieve up to 25X nuclear genome coverage. We provide guidance for read mapping to a reference genome and discuss the limitations of relying on small fragments for SNP genotyping and <jats:italic>de novo<\/jats:italic> genome assembly. This protocol opens the door to broader-scale genetic and phylogenetic analysis of historical specimens, contributing to a deeper understanding of evolutionary trends and adaptation in response to changing environments.<\/jats:p>","DOI":"10.1371\/journal.pone.0296491","type":"journal-article","created":{"date-parts":[[2024,1,2]],"date-time":"2024-01-02T19:09:47Z","timestamp":1704222587000},"page":"e0296491","update-policy":"https:\/\/doi.org\/10.1371\/journal.pone.corrections_policy","source":"Crossref","is-referenced-by-count":19,"title":["Hot alkaline lysis gDNA extraction from formalin-fixed archival tissues"],"prefix":"10.1371","volume":"19","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-6459-9508","authenticated-orcid":true,"given":"Erin 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Kehlmaier","year":"2019","journal-title":"J Zoolog Syst Evol Res."},{"issue":"1","key":"pone.0296491.ref005","doi-asserted-by":"crossref","first-page":"e0225807","DOI":"10.1371\/journal.pone.0225807","article-title":"Vortex fluidics-mediated DNA rescue from formalin-fixed museum specimens","volume":"15","author":"CA Totoiu","year":"2020","journal-title":"PLoS One"},{"issue":"1755\u20130998.13433","key":"pone.0296491.ref006","article-title":"Successful application of ancient DNA extraction and library construction protocols to museum wet collection specimens","author":"N Straube","year":"2021","journal-title":"Mol Ecol Resour"},{"issue":"6","key":"pone.0296491.ref007","doi-asserted-by":"crossref","first-page":"2130","DOI":"10.1111\/1755-0998.13505","article-title":"Unlocking inaccessible historical genomes preserved in formalin","volume":"22","author":"EE Hahn","year":"2022","journal-title":"Mol Ecol Resour"},{"issue":"2","key":"pone.0296491.ref008","doi-asserted-by":"crossref","first-page":"487","DOI":"10.1111\/1755-0998.13481","article-title":"Genomic library preparation and hybridization capture of formalin-fixed tissues and allozyme supernatant for population genomics and considerations for combining capture- and RADseq-based single nucleotide polymorphism data sets","volume":"22","author":"KA O\u2019Connell","year":"2022","journal-title":"Mol Ecol Resour"},{"key":"pone.0296491.ref009","doi-asserted-by":"crossref","first-page":"81","DOI":"10.1007\/978-1-61779-516-9_11","article-title":"DNA extraction from formalin-fixed material","volume":"840","author":"PF Campos","year":"2012","journal-title":"Methods Mol Biol"},{"issue":"2","key":"pone.0296491.ref010","doi-asserted-by":"crossref","first-page":"375","DOI":"10.1007\/s10592-021-01424-4","article-title":"Contextualizing enigmatic extinctions using genomic DNA from fluid-preserved museum specimens of Desmognathus salamanders","volume":"23","author":"RA Pyron","year":"2022","journal-title":"Conserv Genet"},{"key":"pone.0296491.ref011","first-page":"2023.07.26.550239","article-title":"Century-old chromatin architecture preserved with formaldehyde","author":"EE Hahn","year":"2023","journal-title":"bioRxiv"}],"container-title":["PLOS 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