Maureen Purcell 2017 tab delimited file Reston, VA U.S. Geological Survey ScienceBase https://doi.org/10.5066/F7FN14QN Maureen Purcell Rachel Powers Bonnie Besijn Paul Hershberger 2017 tab delimited file Journal of Aquatic Animal Health American Fisheries Society https://doi.org/10.1080/08997659.2017.1365780 The data are used to support the development and validation of two quantitative polymerase chain reaction (qPCR) assays to detect the parasite Nanophyetus salmincola DNA in water samples and fish and snail tissues. The data link to a series of experiments that are described in the publications. Experiment 1 defines the linearity and detection limits of the assays. Experiment 2 assesses the utility of the assays to detect N. salminicola waterborne cercaria in one liter volumes of water. Experiment 4 assesses the detection of N. salminicola waterborne cercaria in small volumes (50 microliters). Experiments 5 and 6 assesses the within and between run repeatability of the assays. Experiment 7 assesses the diagnostic specificity of the assays in a fish population known to be negative for the parasite. Experiment 8 assesses the diagnostic specificity of the assays in a fish population known to be positive for the parasite. Experiment 9 assesses the diagnostic accuracy of the assays in fish artificially exposed to the parasite. Experiment 10 assesses the ability of the assay to detect the parasite in the intermediate snail host (Juga spp.). Experiment 11 assesses the ability of the assay to detect the parasite in the one liter volumes of river water collected at sites where the intermediate snail host is either present or absent. The data are used to support the development and validation of two quantitative polymerase chain reaction (qPCR) assays to detect N. salmincola DNA in water samples and fish and snail tissues. 2015 2017 publication date Complete Not planned USGS Thesaurus Disease research DNA probe analysis USGS Metadata Identifier USGS:59371332e4b0f6c2d0d89a0d USGS Thesaurus Washington none none Maureen Purcell Western Fisheries Research Center mailing and physical

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Seattle WA 98034 USA 206-526-2052 mpurcell@usgs.gov USGS Biocomplexity Thesaurus Fishes Integrated Taxonomic Information System (ITIS) 2017 ONLINE_REFERENCE Washington, D.C. Integrated Taxonomic Information System (ITIS) http://itis.gov Maureen Purcell Western Fisheries Research Center mailing and physical

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Seattle WA 98034 USA 206-526-2052 mpurcell@ugsg.gov expert advice;;genetic analysis Organism identification is accurate and no challenges encountered Fish were identified to the level of species (Oncorhynchus mykiss) Snails were identified to the level of genus (Juga) Digenean trematodes were identified to the level of species (Nanophyetus salmincola) Kingdom Animalia Subkingdom Bilateria Infrakingdom Protostomia Superphylum Platyzoa Phylum Platyhelminthes Subphylum Neodermata Class Trematoda Subclass Digenea Order Plagiorchiida Suborder Troglotremata Family Nanophyetidae Genus Nanophyetus Species Nanophyetus salminicola All data have been cross-checked and are accurate All data have been checked for duplication and omission and none were found. Data set is considered complete for the information presented, as described in the abstract. Users are advised to read the rest of the metadata record carefully for additional details. No formal positional accuracy tests were conducted No formal positional accuracy tests were conducted Software associated with Viia7 (Life Technologies Inc.) 2017 Lab Real time PCR reactions were conducted using the ViiA 7 real-time PCR system (Life Technologies). The standard reaction protocol contained 12 µl of real-time PCR master mix (Taqman Gene Expression master mix; Life Technologies) with 900 nM of each primer and 200 nM of the probe and 5 µl of DNA template. For some tests, an exogenous internal positive control (IPC; Life Technologies; following manufacturer instructions) was included with the N. salmincola primer and probes as a multiplex reaction to test for PCR inhibition. The multiplex reactions were conducted as described above but in a final 24 µl reaction volume. Assay performance was compared using the Taqman Gene Expression Master Mix and the Taqman Environmental Master Mix (both obtained from Life Technologies Inc.). The default protocol was the singleplex (12 µl reaction) protocol lacking the IPC. Taqman Gene Expression master mix was used for detecting Nanophyetus salmincola DNA in snails and fish tissues as well as from isolated cercariae while the Environmental Master Mix was used for detection of cercariae in settled (environmental) water samples. The PCR reactions were subjected to one cycle of 50°C for 2 min, one cycle of 95°C for 10 min, and 40 cycles of 95°C for 15 sec and 60°C for 1 min. Tests were interpreted as positive if sigmodal amplifications curves were generated in < 40 cycles in both technical replicates. Purcell_Nanophyetus_Data_Release_2017 Raw data that supports the development and validation of quantitative PCR for Nanophyetus salmincola Maureen Purcell Unique_Experiment_ID A unique, arbitrary number to track each experiment. Producer defined 1 11 individual experiment number (arbitrary value) Unique_Sample_ID An arbitrary numeric identifier of individual samples within an experiment. Producer defined These are arbitrary numerical values that allowed us to track individuals samples Sample_Site A text definition of where the sample was collected Producer defined na non applicable Producer defined Jenkins Creek, WA a sample collected from Jenkins Creek, WA Producer defined Marblemount Hatchery, WA a sample collected from Marblemount Hatchery, WA Producer defined Nisqually River, WA a sample collected from Nisqually River, WA Producer defined Nordland, WA Samples generated during an experiment conducted at the Marrowstone Marine Field Station, Nordland, WA Producer defined Soos Creek Hatchery, WA A sample collected at Soos Creek Hatchery, WA Producer defined Latitude The geographic latitude Google Maps latitude geographic coordinates Longitude The geographic latitude Google Map longitude geographic longitude Parasite_Organism Presence of a parasite in the sample Producer defined na non-applicable (no parasite in the sample) Producer defined Nanophyetus salmincola The parasite Nanophyetus salmincola was present in the sample Producer defined Host_Organism The name of the host animal associated with the parasite Producer defined Juga spp. Species within the genus Juga - a type of aquatic snail Producer defined Oncorhynchus mykiss The anadromous form of Oncorhynchus mykiss commonly called steelhead trout Producer defined Sample_Type A description of the type of sample tested Producer defined Bacterial Plasmid The DNA tested was from an artificially produced plasmid that encodes the quantitative PCR target region Producer defined Tissue Sample was a tissue (either fish or snail) Producer defined water Sample was a large volume of water Producer defined waterborne cercaria Sample was a waterborne cercaria from Nanophyetus salmincola Producer defined Treatment A brief description of the type treatment or classification of the sample Producer defined Control not exposed to the parasite Nanophyetus salmincola Producer defined Exposed to N. salmincola exposed to the parasite Nanophyetus salmincola Producer defined Juga spp. observed at the sample site Water samples were taken from sample sites where Juga spp. was observed Producer defined No Juga spp. observed at the sample site Water samples were taken from sample sites where Juga spp. was not observed Producer defined na not applicable Producer defined Negative for visible N. salminocola Samples were negative for visible N. salminocola by microscopy Producer defined Positive for visible N. salminicola Samples were positive for visible N. salminocola by microscopy Producer defined Template Template material used in the quantitative polymerase chain reaction (qPCR) reaction Producer defined DNA genomic DNA used from a variety of sources (tissues, water or parasite cercaria) Producer defined Plasmid DNA DNA was from an artificially produced plasmid Producer defined DNA_concentration_ng_per_microliter The DNA concentration of the template in nanogram per microliter volumes; samples labeled 'nd' means that no readings were taken Producer defined 53.5 2428.9 nanogram per microliter Quantitative_PCR_Assay_Name The name of the quantitative PCR assay Producer defined Nano Zen Nanophyetus salmincola quantitative PCR assay that utilizes a Zen internally quenched probe Producer defined Nano LNA Nanophyetus salmincola quantitative PCR assay that utilizes a locked nucleic acid probe Producer defined PCR_Reaction_Type Whether the run was run a singleplex (a single qPCR assay per tube) or a multiplex (two qPCR assays per tube) Producer defined Singleplex Single qPCR assay per tube Producer defined Multiplex two qPCR assays per tube Producer defined Extraction_Method Refers to whether this a direct extraction of tissue or cercaria or whether this extraction of water (one liter volume) Producer defined direct Direct extraction of a tissue, cercaria or other sample Producer defined water extraction of the settled contents of one liter of water Producer defined Known_Nanophyetus_cercaria_number This defines whether the sample contained a known number of cercaria; samples labeled na (not-applicable) did not have enumerated cercaria numbers Producer defined 0 512 Producer defined Known_DNA_Copy_Number This defines whether the sample contained a known number of N. salmincola DNA copies; samples labeled na (not-applicable) did not have enumerated copy numbers Producer defined 1.8 82000000 DNA copies Cycle_Threshold_CT_Replicate_1 The quantitative PCR cycle threshold (CT) value that is used to estimate the DNA copy number in technical replicate well 1; samples labeled undetermined do not have a value determined. Producer defined 12.8 39.5 cycle threshold values undetermined no cycle threshold value was determined (sample is below detection limit) producer defined Cycle_Threshold_CT_Replicate_2 The quantitative PCR cycle threshold (CT) value that is used to estimate the DNA copy number in technical replicate well 2; samples labeled undetermined do not have a value determined; samples labeled as 'na' did not have a second replicate well run Producer defined 12.8 38.22 cycle threshold undetermined no cycle threshold value determined producer defined na no second replicate well was conducted producer defined PCR_estimated_copy_number_per_reaction The estimated copy number per reaction for each sample that was determined based on the cycle threshold values producer defined 0 94000000 DNA copies per reaction na not applicable because it was not calculated producer defined Dilution_Factor The reciprocal dilution of the sample that was run in the quantitative PCR; estimated copy number multiplied by the reciprocal dilution factor equates to the total copies in the sample Producer defined 1 10000 reciprocal dilution factor na not applicable; no dilution is relevant to this sample Producer defined U.S. Geological Survey - ScienceBase mailing and physical

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Denver CO 80225 USA 1-888-275-8747 sciencebase@usgs.gov Unless otherwise stated, all data, metadata and related materials are considered to satisfy the quality standards relative to the purpose for which the data were collected. Although these data and associated metadata have been reviewed for accuracy and completeness and approved for release by the U.S. Geological Survey (USGS), no warranty expressed or implied is made regarding the display or utility of the data for other purposes, nor on all computer systems, nor shall the act of distribution constitute any such warranty. software that can read tab-delimited files 20200830 Maureen Purcell Western Fisheries Research Center Supervisory Research Microbiologist mailing and physical

6505 NE 65th ST

Seattle WA 98034 USA 206-526-2052 mpurcell@usgs.gov FGDC Biological Data Profile of the Content Standard for Digital Geospatial Metadata FGDC-STD-001.1-1999