Katarzyna Przybyla-Kelly Ashley Spoljaric Muruleedhara Byappanahalli Mary Anne Evans 20250331 spreadsheet Reston, VA U.S. Geological Survey Katarzyna Przybyla-Kelly, USGS Great Lakes Science Center, https://orcid.org/0000-0001-9168-3545; Ashley Spoljaric, Michigan State University contractor to USGS Great Lakes Science Center, https://orcid.org/0000-0001-6262-030X; Muruleedhara Byappanahalli, USGS Great Lakes Science Center, https://orcid.org/0000-0001-5376-597X; Mary Anne Evans, USGS Great Lakes Science Center, https://orcid.org/0000-0002-1627-7210 https://doi.org/10.5066/P1S9XBT8 This dataset is associated with an examination of environmental DNA (eDNA) from the invasive round goby (Neogobius melanostomus) in parallel with visual surveys of fish count and size conducted by scuba divers in four Great Lakes: Michigan, Huron, Erie, and Ontario. Round goby inhabits benthic areas, and it is known to have direct contact with the bottom substrate as it feeds on dreissenid mussels. Considering this, eDNA samples were derived from multiple substrates: lake bottom water, sediment, and benthic algae; each manually collected by scuba divers from 3 or 6-meter depths and eight distinct transects. eDNA samples were analyzed by droplet digital PCR (ddPCR) and results are expressed as DNA Copy Numbers (CN) per 1 mL of water or per 1 gram of wet weight of sediment or benthic algae. All eDNA samples for this dataset were collected alongside a larger body of work conducted in 2022 (https://doi.org/10.5066/P13JDUMH) and relate to multiple years of work at these stations that includes: algal and dreissenid mussel biomass, water quality assessments, and diver observations of dreissenid mussels, round gobies, benthic substrate, and benthic algal cover. We refer to the benthic algae also as the 'Cladophora community' and 'submerged aquatic vegetation (SAV)' in other published project data, which were collected starting in 2018 (Great Lakes Science Center, 2018). These data were collected to compare detection of an aquatic organism via eDNA and visual surveys. The ddPCR method was chosen not only to maximize the detection likelihood of round goby DNA in water, sediment, and benthic algae samples, but also to decrease the possibility of inhibition affecting the results since eDNA is extracted from solid substrates. It should be noted, sampling locations were not randomized, therefore, round goby counts should not be extrapolated to infer total round goby population per lake. Nonetheless, this research will help resource managers to better understand the use of eDNA in population assessments compared to traditional surveys. 20220608 20220825 ground condition Complete None planned Lake Michigan, Lake Huron, Lake Erie, and Lake Ontario -87.8906 -77.2119 45.7369 41.6565 ISO 19115 Topic Category biota environment inlandWaters USGS Thesaurus field sampling algal blooms environmental DNA nuisance species algae freshwater ecosystems benthic ecosystems polymerase chain reaction nonindigenous species fish invasive species USGS Metadata Identifier USGS:67c87fd2d34ea599a3b9ac4f Getty Thesaurus of Geographic Names Great Lakes Region Common geographic areas Lake Michigan Lake Erie Lake Huron Lake Ontario No access constraints are associated with these data. No use constraints are associated with these data. Katarzyna Przybyla-Kelly USGS Great Lakes Science Center Biologist mailing and physical

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Chesterton Indiana 46304 USA 219-926-8336 kprzybyla-kelly@usgs.gov The U.S. Environmental Protection Agency granted funding to the U.S. Geological Survey under the Great Lakes Restoration Initiative for data collection and analysis. QX Manager 1.2 Standard Edition was used to obtain ddPCR data. Madeleine M Giordano Shelby L Eagan Alexander E Kacher Megan E Lewan Jessica C Oswald Katarzyna Przybyla-Kelly Dawn A Shively Ashley M Spoljaric Mary A Evans 2024 dataset https://www.sciencebase.gov U.S. Geological Survey https://doi.org/10.5066/p13jdumh Thorough quality assurance practices were employed in laboratory and field protocols to collect the data, ensuring the accuracy of values in datasets. All technicians collecting data undergo rigorous training to adhere to SOPs. All glassware and sampling equipment was either single-use or bleach-sterilized to avoid cross-contamination. Appropriate positive and negative controls were included in all ddPCR assays; none of the 10 extractions blanks exhibited positive amplification. A single extraction blank was included with every set of 11 DNA extractions; all blanks tested negative in ddPCR runs. Datasets were checked for errors, including duplication or omission. For ddPCR, control wells were inspected to determine the assay was performing correctly, only wells with >10,000 droplets were used, and thresholds were manually set per recommendations of the manufacturer. 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. Station locations were established by GPS, with a horizontal precision of 0.00001 degrees (~1.1 m) latitude and longitude. Due to wind-induced movement or boat anchoring, the horizontal precision for sampling locations was thus lower than GPS precision, averaging approximately 30 m. Water column depth was measured by a GPS depth sounder. All depths relative to the water surface can be impacted by waves and have decreased accuracy during rough conditions. Sample and measurement depths relative to the sediment-water interface (for benthic samples) were determined by diver equipment placement and have an accuracy of plus or minus 0.03 meters. Katy E. Klymus Dannise V. Ruiz Ramos Nathan L. Thompson Catherine A. Richter 20201105 publication Journal of Visualized Experiments issue 165 n/a MyJove Corporation https://doi.org/10.3791/61825 Digital and/or Hardcopy 20201105 publication date Klymus et al., 2020 Publication used for IPC and duplexing in STEP 4 - Molecular work. Kasia J. Przybyla-Kelly Ashley M. Spoljaric Meredith B. Nevers 20230106 publication Fishes vol. 8, issue 1 n/a MDPI AG ppg. 41 https://doi.org/10.3390/fishes8010041 Digital and/or Hardcopy 20230106 publication date Kelly et al., 2023 Publication used to reference a source of modified round goby molecular assay used in ddPCR analysis in STEP 4 - Molecular work Lucas M. Nathan Megan Simmons Benjamin J. Wegleitner Christopher L. Jerde Andrew R. Mahon 20141023 publication Environmental Science and Technology vol. 48, issue 21 n/a American Chemical Society (ACS) ppg. 12800-12806 https://doi.org/10.1021/es5034052 Digital and/or Hardcopy 20141023 publication date Nathan et al., 2014 Publication used to reference a source of original round goby molecular assay used in ddPCR analysis in STEP 4 - Molecular work Madeleine M Giordano Shelby L Eagan Alexander E Kacher Megan E Lewan Jessica C Oswald Katarzyna Przybyla-Kelly Dawn A Shively Ashley M Spoljaric Mary A Evans 2024 dataset https://www.sciencebase.gov U.S. Geological Survey https://doi.org/10.5066/p13jdumh Digital and/or Hardcopy 2024 publication date Giordano et al., 2024 Larger dataset providing additional data and source of some data in Step 1 and 2. STEP 1 Collection sites and regime Samples were collected at 8 transects across 4 Great Lakes, Michigan, Huron, Erie, and Ontario; details on sampling locations are provided in the Locations2022 table of the project data release (Giordano et al., 2024: https://doi.org/10.5066/P13JDUMH). All eDNA samples (water, sediment, algae) were collected at a single station (nominal depth of 6 m); on two occasions benthic algae samples were collected at additional stations (nominal depth of 3 m) due to unavailability of algae at the 6 m station. The study period was from 6/8/22 through 8/25/22. Field collections - Water samples Bottom water samples were collected by scuba divers using 2 L bleach-sterilized Nalgene bottles; empty bottles had a 3 lb scuba weight taped to the outside to facilitate transport to the lake bottom. To collect water, divers swam away from the boat's anchor, slowly and above the lake's bottom to prevent sediment and organic material resuspension. Once arrived at the sampling location, usually near large areas covered with dreissenid mussels and benthic algae, divers removed the lid from the bottle slowly while holding the bottle upside down. The bottle was then gently turned upward allowing it to slowly fill with water, capped once filled, and returned to the surface. Due to logistical constraints, replicate water samples were limited to two visits (MWA and EEP July). Water samples were taken from about 10 cm above lake bottom substrate. After retrieving samples on the boat deck, bottom water samples were transferred to a cooler and kept on ice until filtration. Maximum holding time for water samples was 24 hours. Field collections - Benthic samples: algae and sediment Benthic algae were collected by scuba divers by hand pulling attached filaments and placing them in a prelabeled Nitex 500 microliter mesh bag. All types of benthic algae (i.e. Cladophora sp, Chara sp, stalked diatoms, non-branching filamentous green algae) present were collected as a combined bulk sample. After algae samples were returned to the boat, two subsamples, with a minimum size of a quarter coin, were removed from the bulk sample with gloved hands, placed in a 7 oz wire-top plastic bag, and kept in a cooler on dry ice until transported back to the laboratory, where they were kept frozen at -20 degrees C until processed. Lake bottom sediment samples were collected using sterile 90 mL urine cup containers with two replicates at each site. Underwater, divers removed the lid from the sampling cup and collected a sample by sliding the cup over or scooping the sediment; cups were capped and placed in a zip-top freezer bag underwater. In places where the bottom substrate was dominated by bedrock, large boulders, cobble, and/or with little amount of finer sediment, the sampling cup was slid around in crevices and around rocks. In places where bottom substrate was composed mainly of shell hash, samples were taken in multiple areas where sediment was visible, but frequently contained shell fragments. After returning to the boat, sediment samples were placed in individual wire-top plastic bags and kept in a cooler on dry ice until transported back to the laboratory, where they were kept frozen at -20 degrees C until processed. More analyses of algae and sediment samples (i.e. tissue nutrient content) are included in the BenthicChem2022 table of the project data release (Giordano et al., 2024: https://doi.org/10.5066/P13JDUMH). Giordano et al., 2024 2022 STEP 2 – Diver Observations and Data Processing Before collection of benthic samples, divers performed visual assessments encompassing a 6-10 m radius around the anchor while swimming close to, but not disturbing or digging into the bottom. Amongst other observations, divers assessed the abundance and size of round goby fish present in the sampling area; each diver recorded these observations on their personal dive slate. Upon returning to the boat, these observations were recorded on a separate survey form. If the overall observations differed between the divers' reports, they were discussed, and one consensus response was recorded. Additional observations collected by divers (i.e. substrate characteristics, SAV and dreissenid mussels coverage, etc.) are included in the DiverObs2022 table of the project data release (Giordano et al., 2024: https://doi.org/10.5066/P13JDUMH). Diver observations of round goby categorical abundance (GobyAbundance), round goby count for stations where round gobies were abundant (GobyCount), and minimum and maximum observed round goby lengths (GobySizeMin and GobySizeMax, respectively) were extracted from the DiverObs2022 table of the project data release (Giordano et al., 2024: https://doi.org/10.5066/P13JDUMH). To facilitate comparison to ddPCR data, categorical abundance (GobyAbundance) data for the "moderate" abundance category were converted to numerical counts by taking the midpoint (8 fish/m2) of the range ("moderate"= 6-10 fish/m2) unless more descriptive notes were recorded by the divers. LengthMid was derived from LengthMin and LengthMax, please see column definitions for these variables. Giordano et al., 2024 2022 STEP 3 - Laboratory processing of water, benthic algae, and sediment samples Water Filtration Water filtration took place the same day, within about 6 hours of collection, or the next day (within 24 hours of collection). For most samples, 1 L of water was filtered, however, due to differences in water turbidity at various sites, volume of water filtered ranged between 950 - 2100 ml; 2 or even 3 filters were sometimes used per sample due to frequent clogging. Filtration was carried out using a peristaltic pump (Masterflex L/S Portable Sampling Pump), and sterile disposable 250 mL volume capacity filtration cups (Thermo Scientific, Catalog No.09-740-30J) equipped with a 0.2 microliter cellulose nitrate filter set up on a triple-place filtration manifold. The technician performing filtration used gloved hands and frequently wiped with a bleach wipe. Before every pour of the water sample, the bottle was gently inverted five times, and water was poured to the 250 mL line of the filtration cup; volume filtered was recorded in a notebook. Once filtration was completed, the filter was removed from the base using two bleach-sterilized tweezers, folded in half, then into quarters, then eighths (3 folds) and placed in a 5 mL labeled sterile snap cap tube. Tubes containing filters were kept in a wire-top plastic bag on dry ice until transported to the laboratory where they were kept frozen at -20 degrees C until processed. Sediment processing Sediment samples were thawed overnight at 4 degrees C. The content of each urine cup was then emptied into a 14 oz wire-top plastic bag and the descriptive notes characterizing the sediment were documented. For elutriation, 100 mL of Phosphate Buffer Solution (PBS) was added to the wire-top plastic bag containing sediment; if the sediment sample contained overlaying water from the sampling site, the volume of PBS was adjusted accordingly so that total volume of liquid used was 100 mL. Wire-top plastic bags were then closed tightly and the contents were vigorously shaken for two minutes. The sediment was left to settle for <10 seconds, the entire elutriate was transferred to a 200 mL bleach-sterilized plastic bottle, and then the sediment was weighed on a balance to obtain its wet weight. Elutriate was then centrifuged at 8000 RPM (10,017 x g) for 15 minutes; the majority of the supernatant was discarded. The pellet along with a small amount of liquid, was transferred into a sterile 7 mL pre-weighed tube using sterile disposable spatulas and sterile 5 mL pipettes to dislodge and transfer the pellet. All tubes were centrifuged again at 8000 RPM for 15 minutes, supernatant was discarded, and the pellet weight was recorded before placing it at -80 degrees C. Benthic algae processing Benthic algae samples were thawed overnight in 4 degrees C. The wet weight of algae was taken and notes were made on algae type present and its condition. For elutriation, 20 mL of sterile PBS was added straight into the wire-top plastic bag containing algae. Each wire-top plastic bag was then closed tightly and the content was vigorously shaken for two minutes. The entire elutriate was then transferred to a 50 mL sterile centrifuge tube and centrifuged at 8000 RPM (10,017 x g) for 15 minutes, the majority of the supernatant was discarded. The pellet along with a small amount of liquid, was transferred into a sterile 7 mL pre-weighed tube; using sterile disposable spatulas and sterile 5 mL pipettes to dislodge and transfer the pellet. All tubes were centrifuged again at 8000 RPM for 15 minutes, supernatant was discarded, and the pellet weight was recorded before placing it at -80 degrees C. 2022 STEP 4 - Molecular work DNA extractions For water samples, DNA was extracted from filters using the DNeasy Power Soil Pro DNA extraction kit (Qiagen, Carlsbad, CA, USA). If multiple filters were used for the same sample, they were initially processed separately but loaded onto the same MB Spin Column, resulting in a single DNA extract per sample. Frozen filters were first thawed at 4 degrees C, unfolded inside a sterile petri dish using bleach-sterilized tweezers, then rolled with the sample facing inward and placed inside UV-sterilized 7 mL tube containing beads; prior to that, beads were transferred from the original PowerBead Pro tubes to the 7 mL tubes to allow for more space for the filter during the lysis step. For sediment and algae samples, DNA was extracted from pellets using the DNeasy Power Soil Pro DNA extraction kit. A 500 plus or minus 50 mg subsample was used for larger pellets; if a pellet's weight was less than 500 mg, it was used entirely. Exact pellets' weights used for DNA extractions were recorded for later normalization purpose. Pellets were first thawed at 4 degrees C, homogenized with a sterile spatula inside the tube, and a subsample was placed into a new clean tube; single-use spatulas and wooden sticks were used to transfer the pellet. Once the lysis buffer was added to the weighed pellet, a homogeneous slurry was made by aspirating a 1 mL pipette multiple times; the resulting slurry was then transferred into a PowerBead Pro tube. DNA extractions followed the manufacturer's protocol aside from the following changes: (1) after adding the lysis buffer to filters or pellets and prior to the 10 min-vortexing step, tubes were placed into a pre-heated thermal shaker (300 rpm) and incubated for 15 min at 65 degrees C; and (2) DNA was eluted twice using 50 microliters of elution buffer (C6). All DNA extracts were stored at -80 degrees C until used for ddPCR. ddPCR DNA was analyzed in duplicate wells (technical replicates) and quantified using the QX200 AutoDG Droplet Digital PCR System (Bio-Rad, Hercules, CA, USA). Modified from Nathan et al., 2014 and optimized for ddPCR, the following primer and probe set was used: GobyCOI-F2d: 5'- CTTCTGGCCTCCTCTGGTGTTG-3', GobyCOI-R2d: 5'-CCCTAGAATTGAGGAAATGCCGG-3', and Affinity Plus Probe 5'-6-FAM-CAGG+CAACTT+GGC+ACAT+GCAG-3' BHQ-1 (Integrated DNA Technologies, Coralville, IA, USA) (Kelly et al., 2023). A second assay, Hem-T, targeting an unrelated species (mouse) using a different reporter dye (HEX) was used as an internal positive control (IPC) and duplexed in all wells to assess the presence of inhibition (Klymus et al., 2020). Synthetic mouse DNA (gBlock Gene Fragment, Integrated DNA Technologies, Coralville, IA, USA) in the wells containing samples amplified at expected levels similar to the amplification of the mouse DNA in the no-template control (NTC; PCR-grade water) well indicating little to no inhibition was present in the extracted DNA. Each 25 microliters reaction contained: 12.5 microliters of ddPCR Supermix for Probes (no dUTP), 5 microliters template DNA, 1.5 microliters Hem-T DNA, PCR-grade water, and a final concentration of 900 nanomolar (nM) of each primer and 250 nM of each probe. Next, 22 microliters of each reaction was pipetted into DG32 cartridges, which were inserted into the Automated Droplet Generator (AutoDG). After droplet generation, each plate was sealed by heat bonding and contained one well of NTC with IPC and a positive control, diluted DNA extracted from round goby tissue, to assess for contamination and successful target amplification, respectively. Droplets were cycled in a Bio-Rad C1000 Connect thermal cycler with a 96-deep well reaction module (Bio-Rad, Hercules, CA, USA). Thermal conditions for cycling droplets were as follows: 95 degrees C for 10 min, 45 cycles of 95 degrees C for 30 s and 60 degrees C for 1 min, and 1 cycle of 98 degrees C for 10 min with a final 30 min hold at 12 degrees C. After cycling, droplets were streamed on the QX200 Droplet Reader (Bio-Rad,Hercules, CA, USA) in which the number of fluorescent droplets determine round goby concentration after manually setting the threshold in Bio-Rad's QX Manager 1.2 Standard Edition software. The software provided the copies/microliters (CN/microliters) for each reaction, but final concentrations were calculated using the amount of DNA template and PCR reaction. ddPCR results were normalized and expressed as Copy Numbers per milliliter, CN/mL (for water samples) and CN/g of wet weight (for sediment and algae samples). For water samples, to obtain CN/mL of water, initial copies/microliters (CN/microliter) were multiplied by the total volume of PCR reaction, 25 microliters, divided by 5 microliters of the DNA template volume, multiplied by 100 microliters of DNA extraction eluate, and divided by the volume of water filtered. For sediment and algae samples, to obtain CN/g, initial copies/ microliters (CN/microliters) were multiplied by the total volume of PCR reaction, 25 microliters, divided by 5 microliters of the DNA template volume, multiplied by 100 microliters of DNA extraction eluate, divided by sediment/algae weight, multiplied by total pellet weight, and divided by the sub-sample of pellet weight used for DNA extractions. Kelly et al., 2023 Klymus et al., 2020 Nathan et al., 2014 2022 USGS Great Lakes Science Center mailing and physical

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Chesterton IN 46360 219-926-8336 Rgoby_eDNA_ddPCR.csv Round goby eDNA concentrations from water, algae, and sediment samples by ddPCR Producer Defined Date Collection date. Dates are written in year-month-day (yyyy-mm-dd) format Producer Defined 2022-06-08 2022-08-25 Lake Name of the Great Lake where sampling occurred Producer Defined Erie Lake Erie Producer defined Ontario Lake Ontario Producer defined Huron Lake Huron Producer defined Michigan Lake Michigan Producer defined Transect Three-character string indicating which lake and location was sampled Producer Defined Transect codes: EDE, EEP, MWA, MSB, HAL, HHB, OOL, and OIR, correspond to location names. First letter in the code corresponds to Great Lake, i.e. M=Michigan, E=Erie, H=Huron, O=Ontario. Second and third letters correspond to port or transect name, i.e. DE=Dunkirk East; EP=Erie Pennsylvania; WA=Waukegan; SB=Sleeping Bear; AL=Alpena; HB=Hammond Bay; OL=Olcott; IR=Irondequoit. Station Station number associated with targeted depth (meters) where samples were collected within a transect Producer Defined Samplings were targeting 6 meters depth, but on 2 occasions Algae samples were collected at 3 meters depth, as no benthic algae were present at 6 meters. FieldRep Replicated sample for eDNA taken at a site; 1-3 replicates were collected at sampling sites Producer Defined 1 3 TechRep DNA extract from eDNA sample analyzed in replicate Producer Defined 1 2 Water_CN_ml Round goby copy numbers (CN; average of duplicate reactions) of eDNA by droplet digital PCR normalized per milliliter of water Producer Defined <<empty cells>> No data; field replicate was not collected Producer defined 1 25 CN/ml Algae_CN_g Round goby copy numbers (CN; average of duplicate reactions) of eDNA by droplet digital PCR normalized per gram wet weight of algae Producer Defined <<empty cells>> No data; field replicate was not collected Producer defined 0 7265 CN/g Sed_CN_g Round goby copy numbers (CN; average of duplicate reactions) of eDNA by droplet digital PCR normalized per gram wet weight of sediment Producer Defined <<empty cells>> No data; field replicate was not collected Producer defined 0 3238 CN/g Rgoby_fishCount.csv Round goby fish visual observations by scuba divers Producer Defined Date Collection date. Dates are written in year-month-day (yyyy-mm-dd) format Producer Defined 2022-06-08 2022-08-25 Lake Name of the Great Lake where sampling occurred Producer Defined Erie Lake Erie Producer defined Ontario Lake Ontario Producer defined Huron Lake Huron Producer defined Michigan Lake Michigan Producer defined Transect Three-character string indicating which lake and location was sampled Producer Defined Transect codes: EDE, EEP, MWA, MSB, HAL, HHB, OOL, and OIR, correspond to location names. First letter in the code corresponds to Great Lake, i.e. M=Michigan, E=Erie, H=Huron, O=Ontario. Second and third letters correspond to port or transect name, i.e. DE=Dunkirk East; EP=Erie Pennsylvania; WA=Waukegan; SB=Sleeping Bear; AL=Alpena; HB=Hammond Bay; OL=Olcott; IR=Irondequoit. Station Station number associated with targeted depth (meters) where samples were collected within a transect Producer Defined Samplings were targeting 6 meters depth, but on 2 occasions Algae samples were collected at 3 meters depth, as no benthic algae were present at 6 meters. Latitude Latitude in decimal degrees (datum:WGS84) determined by GPS on first site visit Producer Defined 42.17396 45.59494 degrees 0.00001 Longitude Longitude in decimal degrees (datum:WGS84) determined by GPS on first site visit Producer Defined -87.77022 -77.4431 degrees 0.00001 Count Number of round gobies per meter squared Producer Defined 6 30 count per meter squared LengthMin Estimated minimum size of round gobies Producer Defined 1 5 cm LengthMax Estimated maximum size of round gobies Producer Defined 7 17 cm LengthMid Midpoint of LengthMin and LengthMax. Producer Defined 4.5 11 cm U.S. Geological Survey GS ScienceBase mailing address

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Denver CO 80225 United States 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. Any use of trade, firm, or product names is for descriptive purposes only and does not imply endorsement by the U.S. Government. Digital Data https://doi.org/10.5066/P1S9XBT8 No fees are associated with these data. 20250805 Great Lakes Science Center mailing and physical

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Ann Arbor MI 48105 US 734-994-3331 GS_ASK_GLSC@usgs.gov FGDC Biological Data Profile of the Content Standard for Digital Geospatial Metadata FGDC-STD-001.1-1999