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  • This metadata record represents environmental DNA sequence data and metadata barcode file. Seawater and sponge eDNA metabarcoding sampling was conducted at seven coastal locations (Cape Barne, Cape Evans, Cziko Seamount, Granite Harbor Middle, Granite Harbor South, and Turtle Rock) in the Ross Sea to assess spatial eukaryote biodiversity patterns and investigate eDNA signal differences between both substrates. Five replicate 500 mL water samples were collected at each of seven locations within 2 m of the ocean floor using a Niskin bottle. At the same time, five sponge specimens were collected by ROV at a depth range of 18–30 m from three out of the seven locations, thereby enabling sponge and near-bottom water eDNA signal comparison. Further details and laboratory procedures can be found in https://doi.org/10.1002/edn3.500 GET DATA: https://figshare.com/projects/Unveiling_the_Hidden_Diversity_of_Marine_Eukaryotes_in_the_Ross_Sea_A_Comparative_Analysis_of_Seawater_and_Sponge_eDNA_Surveys/186127