Kyle G. George Elizabeth A. Bohuski Jeffrey M. Lorch Anne E. Ballmann 20260121 spreadsheet https://doi.org/10.5066/P14G62B3 These data were collected as part of a research project to compare commonly used DNA extraction methods for subsequent detection of Pseudogymnoascus destructans (Pd), the causative fungus of white-nose syndrome in bats, from bat guano samples, skin swabs, and environmental/sediment swabs. Swab samples were used to evalute DNA extraction protocols based on either a MagMAX™ Total Nucleic Acid Isolation Kit or PrepMan™ Ultra Sample Reagent. Guano samples additionally included the use of a DNeasy PowerLyzer PowerSoil Kit-based DNA extraction protocol. Data_Average_Standard_Curve_v2.csv includes qPCR results (Cq values) from standard curves included on 274 reaction plates. Standards were created from 1:10 dilution series containing 85.296 to 8,529,575 copies of Pd gBlock (i.e., synthetic, double-stranded DNA that contains a near-identical sequence to the intergenic spacer region of the type strain for Pd). A new dilution series was created from a stock aliquot of Pd gBlock at least for each date, but reaction plates processed on the same date may have used the same dilution series. The qPCR methodology for each plate followed that described by Verant et al. (2016). These data were used to create an averaged standard curve, which was used to convert Cq values to copy numbers of target DNA for the remaining datasets in this research project, in which the same qPCR methodology was used. Data_Inoculated_Samples_v2.csv contains qPCR results from a laboratory experiment where guano samples and sediment-coated swabs were inoculated with between 10 and 100,000 Pd conidia and extracted with either a PrepMan, MagMAX, or PowerLyzer-based DNA extraction method. Data_Field-collected_Env_Swabs_v2.csv contains qPCR results from environmental substrate swabs that were collected as part of national Pd surveillance efforts from underground and above-ground bat roosts that were naturally infected with Pd. These environmental swabs were extracted with a PrepMan-based method and qPCR was conducted on both undiluted and a ten-fold dilution of extract from each swab. Data_Bat_Skin_Swabs_v2.csv contains qPCR results from skin swabs collected from bat carcasses that had previously been submitted for diagnostic evaluation of white-nose syndrome infection and were re-sampled for this research project. These skin swabs were extracted with either a PrepMan or MagMAX-based method. The data were collected to identify the optimal methods for DNA extraction of guano, environmental/sediment swabs, and bat skin swabs to support early detection of Pd invasion for national surveillance efforts. 20151216 20220215 observed Complete None planned USGS National Wildlife Health Center, Madison, Wisconsin -89.485617 -89.483128 43.050200 43.047095 ISO 19115 Topic Category biota None PowerLyzer MagMAX PrepMan qPCR disease surveillance USGS Thesaurus white-nose syndrome USGS Metadata Identifier USGS:6616cb47d34e7eb9eb7d69b1 None USGS National Wildlife Health Center, Madison, Wisconsin None Eptesicus fuscus Myotis lucifugus Myotis septentrionalis Perimyotis subflavus Kingdom Animalia Subkingdom Bilateria Infrakingdom Deuterostomia Phylum Chordata Subphylum Vertebrata Infraphylum Gnathostomata Superclass Tetrapoda Class Mammalia Subclass Theria Infraclass Eutheria Order Chiroptera Suborder Yangochiroptera Superfamily Vespertilionoidea Family Vespertilionidae Subfamily Vespertilioninae Tribe Eptesicini Genus Eptesicus Subgenus Eptesicus (Eptesicus) Species Eptesicus fuscus TSN: 180008 Tribe Perimyotini Genus Perimyotis Species Perimyotis subflavus TSN: 947299 Subfamily Myotinae Genus Myotis Subgenus Myotis (Pizonyx) Species Myotis lucifugus TSN: 179988 Species Myotis septentrionalis TSN: 180000 None. Please see 'Distribution Info' for details. None. Users are advised to read the dataset's metadata thoroughly to understand appropriate use and data limitations. Kyle G George U.S. Geological Survey mailing address

6006 Schroeder Road

Madison WI 53711 US 608-270-2463 608-270-2415 kgeorge@usgs.gov We wish to thank the following agencies for their assistance in collecting environmental substrate samples from field sites: Colorado Natural Heritage Program-Colorado State University; Colorado Parks and Wildlife; Georgia Department of Natural Resources; Kansas Department of Wildlife and Parks; Minnesota Department of Natural Resources; Montana Department of Fish, Wildlife and Parks; National Park Service-Fort Laramie National Historic Site; North Carolina Wildlife Resources Commission; South Carolina Department of Natural Resources; South Dakota Department of Game Fish and Parks; US Forest Service-Superior National Forest, MN; US Fish and Wildlife Service Ecological Services-Tulsa, OK; Washington Department of Fish and Wildlife; Wisconsin Department of Natural Resources; and Wyoming Game and Fish Department. Funding for this work was provided by a US Fish and Wildlife Service Interagency Agreement (#4500125334) and the USGS Ecosystems Mission Area. 7500 Fast Real-Time PCR System received twice-annual calibration services. A standard curve was included on each reaction plate to ensure appropriate performance (R2 > 0.95; Efficiency = 90–110%). All data sets were evaluated for outliers, and data checks were conducted in R to ensure expected ranges/entries and sample sizes. Yes Data_Average_Standard_Curve_v2.csv: Only included standard curves that had efficiencies between 90 and 110%. Data_Field-collected_Env_Swabs_v2.csv: Only included environmental swabs in which either or both the undiluted or ten-fold dilution of the PrepMan extract resulted in a Pd detection; negative environmental swabs were excluded from data set. Data_Bat_Skin_Swabs_v2.csv: Samples collected from bat carcasses that were negative for Pd based on initial diagnostic investigation (i.e. negative control samples) were excluded from the data set. Verant ML, Bohuski EA, Lorch JM, Blehert DS. 2016. Optimized methods for total nucleic acid extraction and quantification of the bat white-nose syndrome fungus, Pseudogymnoascus destructans, from swab and environmental samples. Journal of Veterinary Diagnostic Investigation, 28(2): 110–118. Muller LK, Lorch JM, Lindner DL, O’Connor M, Gargas A, Blehert DS. 2013. Bat white-nose syndrome: A real-time TagMan polymerase chain reaction test targeting the intergenic spacer region of Geomyces destructans. Mycologia, 105(2): 253–259. Boyle DG, Boyle DB, Olsen V, Morgan JAT, Hyatt AD. 2004. Rapid quantitative detection of chytridiomycosis (Batrachochytrium dendrobatidis) in amphibian samples using real-time TaqMan PCR assay. Diseases of Aquatic Organisms, 60: 141–148. Hyatt, AD, Boyle DG, Olsen V, et al. 2007. Diagnostic assays and sampling protocols for the detection of Batrachochytrium dendrobatidis. Diseases of Aquatic Organisms, 73: 175–192. Kriger KM, Hines HB, Hyatt AD, Boyle DG, Hero M. 2006. Techniques for detecting chytridiomycosis in wild frogs: comparing histology with real-time TaqMan PCR. Diseases of Aquatic Organisms, 71: 141–148. Forootan A, Sjöback R, Björkman J, Sjögreen B, Linz L, Kubista M. 2017. Methods to determine limit of detection and limit of quantification in quantitative real-time PCR (qPCR). Biomolecular Detection and Quantification, 12: 1–6. 20230522 Data_Inoculated_Samples_v2.csv: Bat guano samples were obtained from three sources: commercial guano fertilizer (FZ; High Nitrogen Bat Guano, Biocontrol Network), guano collected from a bat roost in Oregon (OR) in 2016, and guano obtained from a cave in Tennessee (TN) in 2013. Two pellets, or an equivalent volume, were used for each extraction replicate. Environmental sediment swabs were created by dipping polyester-tipped applicators (Puritan, plastic handles) into 150 µL of sterile water and rolling in a single sediment source until the swab was fully coated. Sediment was sourced from seven geographically distributed bat hibernacula representing caves, mines, and an aqueduct. These hibernacula were located in Connecticut (CT), Illinois (IL), Kentucky (KY), Massachusetts (MA), West Virginia (WV), Wisconsin (WI), and Wyoming (WY). Swabs and guano were placed in tubes used for the first step of each DNA extraction method and were inoculated with either 10, 100, 1,000, 10,000, or 100,000 Pd conidia. Negative extraction controls were spiked with 100 µL of PBST containing no conidia. Three replicates of each conidia concentration were analyzed per sample source and DNA extraction method. Methods for extraction of DNA from guano samples were based on one of three commercial products: PrepMan™ Ultra Sample Preparation Reagent (Applied Biosystems; PrepMan), MagMAX™ Total Nucleic Acid Isolation Kit (Applied Biosystems; MagMAX), or a DNeasy PowerLyzer PowerSoil Kit (Qiagen; PowerLyzer). Comparisons for guano samples were made in two rounds: first by comparing MagMAX to PrepMan, then by comparing PrepMan to PowerLyzer. Thus, conidia used to inoculate each round of samples were harvested from separate cultures. Supernatant from guano extractions were often colored and translucent, which could indicate the presence of humic substances or other PCR inhibitors that would likely interfere with the fluorescence-based assay. Thus, all PrepMan-extractions of guano were diluted ten-fold prior to qPCR. PrepMan and MagMAX-based DNA extraction protocols were compared for detection of Pd from inoculated, sediment-coated swabs. PrepMan extractions of sediment-coated swabs included both undiluted and a ten-fold dilution of extraction product for qPCR. Data_Field-collected_Env_Swabs_v2.csv: Undiluted and a ten-fold dilution of PrepMan extracts were further compared using field-collected environmental swabs acquired from underground and above-ground bat roosts that were naturally infected with Pd. Swabs moistened with 150 µL of sterile water were rolled over substrate in locations where bats were hibernating or in summer roosts. Underground sites were sampled from January–May and included caves, mines, a culvert, and a rock shelter (n = 7 sites), whereas above-ground roosts were sampled from May–July and included bat boxes, bridges, and buildings (n = 19 sites). Samples were processed via the PrepMan-based extraction protocol as stated above for inoculated sediment swabs, and comparisons were made between undiluted and a ten-fold dilution of extract for samples that tested positive for the presence of Pd DNA in at least one of the dilutions. Field-collected swabs were included in this study because they represent a broader range of sediment types and other environmental substrates than were tested in the conidia-spiking experiment. Data_Bat_Skin_Swabs_v2.csv: PrepMan and MagMAX-based protocols were compared for the extraction and detection of Pd DNA from skin swabs collected from bat carcasses that previously had been confirmed positive for the presence of Pd via qPCR. Swabs were collected from nine tricolored (Perimyotis subflavus), five little brown (Myotis lucifugus), three northern long-eared (Myotis septentrionalis), and seven big brown (Eptesicus fuscus) bat carcasses. Carcasses from two additional little brown bat and one tricolored bat carcasses that previously tested negative for Pd served as negative controls. Carcasses had been stored at -20°C since initial diagnostic evaluation and were thawed prior to sample collection. Swabs were moistened with 150 µL of sterile water and rolled 4–6 times across both the muzzle and either the dorsal or ventral side of one wing on each carcass. Only one swab was collected from each side of the wing and was randomly assigned to a DNA extraction method. Extraction protocols followed those outlined for sediment-coated swabs above, except only undiluted extract from the PrepMan protocol was used for qPCR. Quantitative PCR: A 7500 Fast Real-Time PCR System (Applied Biosystems) was used to conduct qPCR on 25-µL reaction volumes as described by Verant et al. (2016). A negative control containing 5 µL of sterile water in place of template DNA was included on each 96-well PCR plate. The threshold was set at 4% of the maximum fluorescence (defined by where the fluorescence plateaued; Verant et al. 2016). Exponential amplification crossing the threshold in fewer than 40 cycles was considered a positive detection for Pd. Quantitative PCR was performed in triplicate for each conidia-inoculated extraction replicate and for each dilution of the PrepMan-extracted sediment swabs. As is typical for Pd surveillance samples, field-collected environmental swabs often were processed in a single replicate for each dilution, but multiple PCR replicates occasionally were performed for confirmatory testing. Additionally, only a single PCR replicate was processed from each skin swab sample. For a subset of inoculated guano samples, cycling conditions were adjusted slightly as follows: standard ramp speed with initial denaturation at 95°C for 3 min followed by 40 cycles of 95°C for 15 s and 60°C for 1 min. These cycling conditions were only used for guano samples extracted via PowerLyzer and the respective batch of PrepMan extractions inoculated with the same harvest of Pd conidia. A qPCR standard curve was generated on each plate using synthetic, double-stranded DNA (gBlock, Integrated DNA Technologies). Two thymine base residues were inserted into the sequence, which was otherwise 100% identical to the intergenic spacer region of the type strain of Pd; these insertions would allow for detection of contamination from the synthetic DNA used for the standard curve through follow-up sequencing of amplicons, if needed. The sequence was as follows: 5’-tct agt cag cct ctc tgg tgg cct ctg cct ctc cgc cat tag tgc cgg tgt agc tgg cgt tac agc ttg ctc ggg ctg cct ctc tag ctg gtt ttg ccg tgg tag ctc acc tac cta gcg agc cgg tgg tgg ctg ctt tgc cg-3’. For initial assays, the gBlock was amplified and the PCR product was cloned into a plasmid, whereas later assays used gBlock directly as template for generating the standard curve. Plasmid standard curves consisted of 5 concentrations of a ten-fold serial dilution ranging from 5x106 to 0.5 fg of plasmid. The gBlock-only templates were included as a 6-point standard curve on each plate with a ten-fold dilution series ranging from 8.5x106 to 85 copies of target DNA. The lowest concentration of standard was run in triplicate on each plate while remaining concentrations were run in duplicate. Results from each plate were considered valid if the R2 value of the standard curve was above 0.95 and the efficiency was between 90% and 110%. Data_Average_Standard_Curve_v2.csv: An averaged standard curve was created by conducting linear regression on gBlock-only standard curves included on 274 reaction plates that followed the same qPCR protocol as stated above (F = 4.254x105, p < 0.0001, R2 = 0.988, y-intercept = 38.683, slope = -3.443, efficiency = 0.952). The equation for this curve was used to convert all Cq values to copies of target DNA (i.e., copy number) detected per reaction: copy number = 〖10〗^(((Cq-yintercept)/slope)). 20230522 Data_Average_Standard_Curve_v2.csv Comma Separated Value (CSV) file containing data. Producer Defined PCR.Plate.Name Plate ID of PCR plate. Producer Defined Plate ID of PCR plate. PCR.Well Well within respective PCR plate Producer Defined Well within respective PCR plate. Rows A–H; Columns 1-12 (i.e., A1 through H12) Cq • PCR cycle number (quantification cycle) at which the sample’s reaction curve intersected the threshold line. Alternatively described as the cycle threshold value (Ct). Producer Defined 13.26 36.38 cycles Qty_Copies • Number of copies of Pd gBlock DNA added to reaction, prepared in a 1:10 dilution series from 8,529,575 copies to 85.29575 copies per reaction. Producer Defined 85.29575 85.29575 Producer defined 852.9575 852.9575 Producer defined 8,529.575 8,529.575 Producer defined 85,295.75 85,295.75 Producer defined 852,957.5 852,957.5 Producer defined 8,529,575 8,529,575 Producer defined Slope Slope of standard curve equation for respective qPCR plate. Producer Defined -3.999 -3.230 R2 R-squared value (coefficient of determination) for standard curve equation of respective qPCR plate. Producer Defined 0.981 1.0 Efficiency Efficiency of standard curve for respective qPCR plate. Producer Defined 90.05 99.95 percentage Date Date PCR was conducted. Producer Defined 20190208 20220215 YYYYMMDD Data_Inoculated_Samples_2.csv Comma Separated Value (CSV) file containing data. Producer Defined PCR.Plate.Name Plate ID of PCR plate Producer Defined Plate ID of PCR plate PCR.Well Well within respective PCR plate. Producer Defined Well within respective PCR plate. A-H and 1-12 Sample.Type Sample type Producer Defined Guano inoculated guano sample Producer defined Env swab inoculated sediment-coated swab Producer defined Extraction.SOP DNA extraction methodology used on sample Producer Defined MagMAX method based on a MagMAX™ Total Nucleic Acid Isolation Kit Producer defined PrepMan method based on PrepMan™ Ultra Sample Preparation Reagent Producer defined PowerLyzer method based on DNeasy PowerLyzer PowerSoil Kit Producer defined Extraction.Date Date that DNA extraction was initiated for sample. Producer Defined 20170119 20171220 YYYYMMDD PCR.Date Date that qPCR was initiated for sample. Producer Defined 20170119 20171221 YYYYMMDD PCR.Protocol Method used for qPCR. Producer Defined Pd PCR this method follows that described by Verant et al. (2016). Producer defined Pd PCR SR this method representations the deviations outlined in the associated publication for a subset of samples (guano samples extracted via PowerLyzer and the respective batch of Prepman extractions inoculated with the same harvest of Pd conidia). Producer defined Cq PCR cycle number (quantification cycle) at which the sample’s reaction curve intersected the threshold line. Alternatively described as the cycle threshold value (Ct). Producer Defined << empty cell >> Results with blank cells represent non-detects. Producer defined 13.28 39.91 cycles Test Test refers to the batch of conidia harvested to create inoculate concentrations. Guano samples extracted via PowerLyzer and a set of Prepman extractions were inoculated with a separate harvest of Pd conidia than the other samples. Producer Defined 1 harvest batch #1 Producer defined 2 harvest batch #2 Producer defined Source The code to identify the source for either the guano or sediment used to create samples. With the exception of commercial fertizlizer (FZ), these codes refer to the state abbreviations from which the guano or sediment originated, in which only a single source site existed in each represented state. Producer Defined FZ Commercial fertilizer Producer defined OR Oregon Producer defined TN Tennessee Producer defined IL Illinois Producer defined KY Kentucky Producer defined MA Massachusetts Producer defined WV West Virginia Producer defined WI Wisconsin Producer defined CT Connecticut Producer defined WY Wyoming Producer defined Conidia Number of Pd conidia included in inoculate for the sample. Producer Defined 0 0 Producer defined 10 10 Producer defined 100 100 Producer defined 1K 1,000 Producer defined 10K 10,000 Producer defined 100K 100,000 Producer defined Rep_Ext Replicate number for the extraction within each treatment group. Three extraction replicates per treatment group. Producer Defined 1 3 Rep_PCR Replicate number for each qPCR run for each extraction replicate. Three extraction replicates per extraction. Producer Defined 1 3 Dilution Dilution of extract used for qPCR. PrepMan-extracted guano samples were run only for a ten-fold dilution, PrepMan-extracted environmental swabs were run both undiluted and for a ten-fold dilution. Producer Defined Undiluted undiluted extract used as template for qPCR. Producer defined Diluted ten-fold dilution of extract used as template for qPCR. Producer defined Det_PCR Indicates whether exponential amplication crossed the threshold during qPCR (i.e., Pd detection). Producer Defined No Pd was not detected for this qPCR replicate Producer defined Yes Pd was detected for this qPCR replicate Producer defined ID_Ext ID that represents the extraction replicate. Created from the combination of the Sample.Type, Extraction.SOP, Test, Source, Conidia, and Rep_Ext fields. Most ID_Ext values have 3 PCR results in the data set, though a subset will have 6 replicates due to both diluted and undiluted extracts have up to 3 PCR results. Producer Defined ID that represents the extraction replicate. Created from the combination of the Sample.Type, Extraction.SOP, Test, Source, Conidia, and Rep_Ext fields. Most ID_Ext values have 3 PCR results in the data set, though a subset will have 6 replicates due to both diluted and undiluted extracts have up to 3 PCR results. ID_Dil ID that represents the extraction replicate and its associated dilution. Created from the combination of the Sample.Type, Extraction.SOP, Test, Source, Conidia, Rep_Ext, and Dilution fields. This field is only relevant for PrepMan-extracted environmental swabs in which both undiluted and a ten-fold dilution of extract is used for qPCR. There may be up to 3 replicates of each ID_Dil in the data set. Producer Defined ID that represents the extraction replicate and its associated dilution. Created from the combination of the Sample.Type, Extraction.SOP, Test, Source, Conidia, Rep_Ext, and Dilution fields. This field is only relevant for PrepMan-extracted environmental swabs in which both undiluted and a ten-fold dilution of extract is used for qPCR. There may be up to 3 replicates of each ID_Dil in the data set. ID_PCR Unique ID associated with each PCR result. Created from the combination of the Sample.Type, Extraction.SOP, Test, Source, Conidia, Rep_Ext, Dilution, and Rep_PCR fields. There should be no duplicates of this ID in the data set. Producer Defined Unique ID associated with each PCR result. Created from the combination of the Sample.Type, Extraction.SOP, Test, Source, Conidia, Rep_Ext, Dilution, and Rep_PCR fields. There should be no duplicates of this ID in the data set. Qty_Copies Copies of target DNA detected from qPCR based on a conversion of the Cq value using the equation for the average standard curve calculated with the Average_Standard_Curve.csv data set. Producer Defined << empty cell >> Results with blank cells represent non-detects. Producer defined 0.44 23937247 number of copies Outlier Indicates whether the result was considered an outlier and subsequently removed from the data set prior to analyses. Producer Defined << empty cell >> not an outlier Producer defined Yes outlier Producer defined Data_Field-collected_Env_Swabs_v2.csv Comma Separated Value (CSV) file containing data. Producer Defined PCR.Plate.Name Plate ID of PCR plate Producer Defined Plate ID of PCR plate PCR.Well Well within respective PCR plate. B-H and 1-12 Producer Defined Well within respective PCR plate. B-H and 1-12 Specimen.Key Unique ID given to each swab sample Producer Defined Unique ID given to each swab sample Sample.Type Indicates sample type. All samples in this data set are environmental swabs Producer Defined Enviro Swab environmental swab of substrate from roost. Producer defined Extraction.Date Date that DNA extraction was performed on the swab sample. Producer Defined 20170403 20210624 YYYYMMDD PCR.Date Date that qPCR was performed on the dilution of extract from the swab sample. Producer Defined 20170403 20210625 YYYYMMDD Cq PCR cycle number (quantification cycle) at which the sample’s reaction curve intersected the threshold line. Alternatively described as the cycle threshold value (Ct). Producer Defined << empty cell >> Results with blank cells represent non-detects. Producer defined 30.12 39.78 cycles Case Number that is unique to the sampling event, which is also unique to each site, with the exception of three sites from MT that were sampled in two separate years. Producer Defined Number that is unique to the sampling event, which is also unique to each site, with the exception of three sites from MT that were sampled in two separate years. State State abbreviation from which the site exists. Producer Defined SC South Carolina Producer defined GA Georgia Producer defined WI Wisconsin Producer defined OK Oklahoma Producer defined NC North Carolina Producer defined KS Kansas Producer defined WY Wyoming Producer defined SD South Dakota Producer defined MN Minnesota Producer defined MT Montana Producer defined CollDate Date that the sample was collected. 20170310 to 20220621; January through July Producer Defined 20170310 20210611 YYYYMMDD Site_Class Field that identifies the type of site sampled. Producer Defined Other-Rock Shelter Site was a rock shelter. Producer defined Cave-undeveloped Site was a cave that was neither used for recreational purposes nor as a show cave. Producer defined Mine-inactive Site is a mine that was no longer active. Producer defined Bat box Site was a bat box(es). Producer defined Bldg/bunker Site was a building or bunker. Producer defined Bridge Site was a bridge. Producer defined Bat box and Bridge Site included both bat box(es) and a bridge. Producer defined Site_Use This field indicates whether the site was being used for either hibernation or as a summer roost during the time of the survey, which also correlates to identification of sites as either underground or above-ground, respectively. Producer Defined Hibernaculum site was an underground site used as a hibernacula. Producer defined Summer Roost site was an above-ground site used as a summer roost. Producer defined Dilution Dilution of extract used for qPCR. Producer Defined Undiluted undiluted extract used as template for qPCR. Producer defined Diluted ten-fold dilution of extract used as template for qPCR. Producer defined Detection Indicates whether exponential amplication crossed the threshold during qPCR (i.e., Pd detection). Producer Defined Detected Pd was detected for this qPCR replicate Producer defined Not Detected Pd was not detected for this qPCR replicate Producer defined Qty_Copies Copies of target DNA detected from qPCR based on a conversion of the Cq value using the equation for the average standard curve calculated with the Average_Standard_Curve.csv data set. Producer Defined << empty cell >> Results with blank cells represent non-detects. Producer defined 0.4803 307.2331 number of copies Data_Bat_Skin_Swabs_v2.csv Comma Separated Value (CSV) file containing data. Producer Defined Bat.ID A unique ID associated with the bat carcass from which the sample was collected. Producer Defined A unique ID associated with the bat carcass from which the sample was collected. Sample.Type Indicates the sample type of the sample. All samples in this data set are skin swabs. Producer Defined Bat Swab skin swab from bat carcass. Producer defined Species Indicates the scientific name (genus and species) of the bat carcass from which the sample was collected. Producer Defined Eptesicus fuscus (Big Brown Bat) Eptesicus fuscus (Big Brown Bat) Producer defined Myotis lucifugus (Little Brown Bat) Myotis lucifugus (Little Brown Bat) Producer defined Myotis septentrionalis (Northern Long-Eared Bat) Myotis septentrionalis (Northern Long-Eared Bat) Producer defined Perimyotis subflavus (Tricolored Bat) Perimyotis subflavus (Tricolored Bat) Producer defined Extraction.SOP Indicates the DNA extraction method that was used on the sample. Producer Defined MagMAX method based on a MagMAX™ Total Nucleic Acid Isolation Kit Producer defined PrepMan method based on PrepMan™ Ultra Sample Preparation Reagent Producer defined Cq PCR cycle number (quantification cycle) at which the sample’s reaction curve intersected the threshold line. Alternatively described as the cycle threshold value (Ct). Producer Defined 18.97 36.75 cycles SpeciesID Four-letter code associated with the bat species from which the sample was collected. Producer Defined EPFU Eptesicus fuscus Producer defined MYLU Myotis lucifugus Producer defined MYSE Myotis septentrionalis Producer defined PESU Perimyotis subflavus Producer defined Qty_Copies Copies of target DNA detected from qPCR based on a conversion of the Cq value using the equation for the average standard curve calculated with the Average_Standard_Curve.csv data set. Producer Defined 3.644 532438 number of copies U.S. Geological Survey GS ScienceBase mailing address

Denver Federal Center, Building 810, Mail Stop 302

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. Digital Data https://doi.org/10.5066/P14G62B3 None 20260121 Kyle G George U.S. Geological Survey mailing address

6006 Schroeder Road

Madison WI 53711 US 608-270-2463 608-270-2415 kgeorge@usgs.gov FGDC Biological Data Profile of the Content Standard for Digital Geospatial Metadata FGDC-STD-001.1-1999