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This project began in 2015, but updates will include legacy data that were collected using the same protocol. The eDNAtlas database consists of four feature classes:The first component (eDNAtlas_East_AGOL_SampleGridAndResults) is a systematically-spaced 1-kilometer grid of potential sample points in streams and rivers throughout the eastern United States. The points in the sampling grid are arrayed along the medium-resolution National Hydrography Dataset Version 2 (NHDPlusV2) and can be used to develop custom eDNA sampling strategies for many purposes. Each sample point has a unique identity code that enables efficient integration of processed eDNA sample results with the species occurrence database.The second component (eDNAtlas_NHDPlusV2_AGOL_ResultsOnly) is a database of georeferenced species occurrence locations based on eDNA field sampling results, which are downloadable by species through dynamic ArcGIS Online (AGOL) mapping tools. The points coincide with the sampling grid and are along the medium-resolution National Hydrography Dataset Version 2 (NHDPlusV2).The earliest eDNA results in the database were collected in 2015 but new samples and results are added annually to the database, which houses thousands of species occurrence records. This feature class includes results from both the western US and the eastern portion of the country.This is the third component (eDNAtlas_NHDPlusHR_AGOL_ResultsOnly)and is a point feature class of all eDNAtlas sample locations that are NOT associated with the eDNAtlas NHDPlusV2 1 km sample grid. These features represent sample locations in smalls streams, lakes, wetlands, springs, and other features that are not represented in the NHDPlusV2 digital data. Many of the sample locations have corresponding features in the NHDPlus High Resolution (NHDPlusHR) dataset. When possible, these sample points are attributed with selected NHDPlusHR attributes. Where no NHDPlusHR feature was present but a valid water sample was collected, field points are represented in this feature class, however no NHDPlus attributes are assigned to the attribute table.This feature class includes results from both the western US and the eastern portion of the country, as well as parts of Alaska.The fourth component (eDNAtlas_West_AGOL_SampleGridAndResults) is a systematically-spaced 1-kilometer grid of potential sample points in streams and rivers throughout the western United States. The points in the sampling grid are arrayed along the medium-resolution National Hydrography Dataset Version 2 (NHDPlusV2) and can be used to develop custom eDNA sampling strategies for many purposes. Each sample point has a unique identity code that enables efficient integration of processed eDNA sample results with the species occurrence database.In the feature class attribute tables, the unique ID for each grid sample point is eDNA_ID. The unique ID for each National Genomics Center for Wildlife and Fish Conservation (NGC) lab result is ID_Tag. There can be more than one ID_Tag per eDNA_ID grid sample point.Detailed metadata available at https://doi.org/10.2737/RDS-2018-0010. 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