Armbrecht, L.Herrando-Pérez, S.Eisenhofer, R.Hallegraeff, G.M.Bolch, C.J.S.Cooper, A.2020-06-242020-06-242020Molecular Ecology Resources, 2020; 20(4):906-9191755-098X1755-0998http://hdl.handle.net/2440/126163Marine sedimentary ancient DNA (sedaDNA) provides a powerful means to reconstruct marine palaeo-communities across the food web. However, currently there are few optimized sedaDNA extraction protocols available to maximize the yield of small DNA fragments typical of ancient DNA (aDNA) across a broad diversity of eukaryotes. We compared seven combinations of sedaDNA extraction treatments and sequencing library preparations using marine sediments collected at a water depth of 104 m off Maria Island, Tasmania, in 2018. These seven methods contrasted frozen versus refrigerated sediment, bead-beating induced cell lysis versus ethylenediaminetetraacetic acid (EDTA) incubation, DNA binding in silica spin columns versus in silica-solution, diluted versus undiluted DNA in shotgun library preparations to test potential inhibition issues during amplification steps, and size-selection of low molecular-weight (LMW) DNA to increase the extraction efficiency of sedaDNA. Maximum efficiency was obtained from frozen sediments subjected to a combination of EDTA incubation and bead-beating, DNA binding in silica-solution, and undiluted DNA in shotgun libraries, across 45 marine eukaryotic taxa. We present an optimized extraction protocol integrating these steps, with an optional post-library LMW size-selection step to retain DNA fragments of ≤500 base pairs. We also describe a stringent bioinformatic filtering approach for metagenomic data and provide a comprehensive list of contaminants as a reference for future sedaDNA studies. The new extraction and data-processing protocol should improve quantitative paleo-monitoring of eukaryotes from marine sediments, as well as other studies relying on the detection of highly fragmented and degraded eukaryote DNA in sediments.en© 2020 John Wiley & Sons Ltdancient DNAdiatomsdinoflagellateshaptophytesMaria IslandmetagenomicsplanktonseafloorTasmaniaJournal article100001904710.1111/1755-0998.131620005319035000012-s2.0-85084488060529299Armbrecht, L. [0000-0002-1213-1257]Herrando-Pérez, S. [0000-0001-6052-6854]Cooper, A. [0000-0002-7738-7851]