Kyle G. George Anne E. Ballmann 20240404 spreadsheet https://doi.org/10.5066/P13RB3ZK Storage-Extraction_Experiment.csv: These data include the qPCR results (Ct values and copy numbers) of DNA extracted from swab samples inoculated with Pd conidia (1,000, 10,000, or 100,000 conidia), stored at various temperatures (ambient temperature, 4°C, or -20°C), with or without RNAlater® preservative, for various durations (3 days, 1 month, or 3 months), and extracted with either a Blood and Tissue Kit-based or PrepMan-based protocol. This data set was used to evaluate the effect of both storage condition and DNA extraction method on Pd DNA recovery from swab samples. LOD_Experiment.csv: These data include qPCR results (Ct values and copy numbers) of DNA extracted from swab samples using either a Blood and Tissue Kit-based or a PrepMan-based protocol following inoculation with a 1:2 dilution series of Pd conidia (4 to 256 conidia). An additional inoculation treatment of 10,000 conidia was also included with this data set. This data set was used to establish and compare the lower limit of detection (in terms of conidia concentration) for the two DNA extraction protocols. The data were collected to identify the optimal methods for sample handling and processing to support early detection of Pd invasion for national surveillance efforts. 20211216 20211217 20220208 20220209 20220519 20220520 20221116 20221117 observed Complete None planned USGS National Wildlife Health Center, Madison, WI, USA -89.4859 -89.4832 43.0463 43.0501 ISO 19115 Topic Category biota None DNA preservation RNAlater® PrepMan™ Ultra Sample Preparation Reagent DNeasy Blood and Tissue Kit temperature USGS Thesaurus white-nose syndrome USGS Metadata Identifier USGS:660ede9fd34ededd8cd825ad None USGS National Wildlife Health Center, Madison, WI, USA 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 Tyler McLaughlin and Amy Haeseler for their assistance in sample processing, and Katherine Richgels for project study design consultation. 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%). Standard curve equations reevaluated in R statistical software to confirm match to instrument output. R statistical software used to convert Cq values to copy numbers (based on standard curve equations) to verify calculations from instrument outputs. Potential outliers evaluated by creating box plots in R. Yes. Treatment groups with too few data points for statistical analyses were excluded from data set, but no data points were excluded from any of the treatment groups included in analyses. George KG, Bohuski EA, Lorch JM, Ballmann AE. In review. Detection Sensitivity for Pseudogymnoascus destructans Differs with DNA Extraction Method and Sample Type: Bat Skin Swabs, Guano, and Environmental Swabs. Molecular Ecology Resources 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. Huebschman JJ, Hoerner SA, White JP, Kaarakka HM, Parise KL, Foster JT. 2019. Detection of Pseudogymnoascus destructans on Wisconsin bats during summer. Journal of Wildlife Diseases, 55(3): 673–677. Shuey MM, Drees KP, Lindner DL, Keim P, Foster JT. 2014. Highly sensitive quantitative PCR for the detection and differentiation of Pseudogymnoascus destructans and other Pseudogymnoascus species. Applied and Environmental Microbiology, 80(5): 1726–1731. 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. Klymus KE, Merkes CM, Allison MJ.(2020). Reporting the limits of detection and quantification for environmental DNA assays. Environmental DNA, 2(3): 271–282. 20211216 Storage-Extraction_Experiment.csv: Recovery of Pd DNA was measured via qPCR following DNA extraction of sterile swabs that had been inoculated with either 1,000, 10,000, or 100,000 Pd conidia. For a subset of samples, 0.5 mL of RNAlater® (Sigma-Aldrich) was added. Samples were stored at either ambient temperature, 4°C, or -20°C in a frost-free freezer for either 3 days, 1 month, or 3 months prior to DNA extraction. A different harvest of Pd conidia was used to inoculate swabs for the 3-day storage duration than either 1 month or 3 months. Extraction methods were based on either PrepMan™ Ultra Sample Preparation Reagent (Applied Biosystems; PrepMan) or a DNeasy 96 Blood and Tissue Kit (Qiagen; B&T Kit). 20211216 LOD_Experiment.csv: To evaluate detection sensitivity based extraction protocols at low concentrations of Pd DNA, swab samples were inoculated with a 1:2 dilution series ranging from 4 to 256 Pd conidia. Additional replicates were inoculated with 10,000 conidia. Samples were extracted using the PrepMan and B&T Kit-based methods as described for the Storage-Extraction_Experiment.csv data. Quantitative PCR: Quantitative PCR was conducted on a 7500 Fast Real-Time PCR System (Applied Biosystems) following previously described methods (Verant et al. 2016, Muller et al. 2013) but with 1x QuantiNova Probe PCR Master Mix (Qiagen). Samples evaluated for DNA recovery after sample storage were run in 25-µL volumes per Verant et al. (2016), with 0.4 µM of each primer, 0.4 µg/µL of BSA, 0.2 µM of probe, and 5 µL of template. A six-point standard curve was included in triplicate on each qPCR plate using a 10-fold dilution series ranging from 85 to 852,958 copies of synthesized gBlocks™ Gene Fragment (gBlock, Integrated DNA Technologies; George et al. in press). For the limit of detection (LOD) analyses, reactions were run in 20-µL volumes with a 0.25x concentration of ROX reference dye to accommodate an optimized methodology for the QuantiNova™ master mix. As such, the standard curve dilution series was adjusted to a range of 34 to 3,411,830 copies of gBlock to incorporate a lower limit of quantification (LOQ = 34 copies), where the coefficient of variation among replicates is ≤ 35% (Forootan et al. 2017). A negative template control containing 5 µL of sterile water was included on all reaction plates. Cycling conditions included 95°C for 2 min followed by 40 cycles of 95°C for 5 s and 60°C for 30 s at a fast ramp speed for all plates. The amplification curve threshold was set at 4% of maximum fluorescence as defined by wells where the fluorescence plateaued (Verant et al. 2016). Accepted results required an R2 value for the standard curve of ≥0.95 and an efficiency between 90% and 110%. A signal that crossed the threshold in fewer than 40 cycles and contained an exponential amplification curve was considered a positive detection for Pd. A single PCR replicate was evaluated per extracted sample, and samples were distributed across reaction plates to ensure plate-to-plate variation did not bias results. Quantities were converted from the quantification cycle (Cq) value to copy number based on the standard curve included on each individual reaction plate: copy number = 〖10〗^(((Cq-yintercept)/slope)). 20221116 LOD_Experiment.csv Comma Separated Value (CSV) file containing data. Producer Defined Treatment Combination of Extraction.SOP and Conidia fields Producer Defined Combination of Extraction.SOP and Conidia fields Extraction.SOP DNA extraction protocol used on sample prior to qPCR. Producer Defined B&T Kit Blood & Tissue Kit-based protocol Producer defined PrepMan PrepMan-based protocol Producer defined Conidia Quantity of conidia included in inoculate for that sample. Producer Defined 10000 10000 Producer defined 256 256 Producer defined 128 128 Producer defined 64 64 Producer defined 32 32 Producer defined 0 0 Producer defined 16 16 Producer defined 8 8 Producer defined 4 4 Producer defined Ct.value • Cycle threshold value, alternatively described as the quantification cycle value (Cq value), produced during qPCR Producer Defined << empty cell >> Samples with blank cells represent non-detects. Producer defined 24.07 39.61 cycles Copies • Copies of target DNA detected from qPCR based on a conversion of the Ct value (Cq value) using the standard curve equation associated with each PCR plate. The values were produced from the qPCR machine output. As such, there will be some slight rounding error when converting the reported Ct values with the reported standard curve equations. Producer Defined << empty cell >> Samples with blank cells represent non-detects. Producer defined 0.4776562 16258.393499 number of copies Detection • Indication of whether there was exponential amplification during qPCR that crossed the threshold in < 40 cycles, which would determine whether Pd DNA was detected from the sample. Producer Defined No there was not exponential amplification within 40 cycles (i.e. there was no Ct value result) Producer defined Yes there was exponential amplification within 40 cycles (i.e. there was a Ct value result) Producer defined PCR_Plate Plate ID of PCR plate. Producer Defined 1 8 yint Y-intercept value of standard curve equation for respective qPCR plate. Producer Defined 37.92934 39.23141 cyces eff Efficiency of standard curve for respective qPCR plate. Producer Defined 0.934600318578839 0.997293782696709 slope Slope of standard curve equation for respective qPCR plate. Producer Defined -3.48929 -3.32843 r2 R-squared value (coefficient of determination) for standard curve equation of respective qPCR plate. Producer Defined 0.9987 0.9997 Storage-Extraction_Experiment.csv Comma Separated Value (CSV) file containing data. Producer Defined PCR_Plate Plate ID of PCR plate. Producer Defined 3 18 TreatmentID Treatment ID number associated with Treatment field. Producer Defined 1 6 Treatment Treatment group. Producer Defined 1: B&T Kit @ AT (RNAlater) Treatment 1: Samples extracted with the Blood and Tissue Kit-based DNA extraction protocol following storage at ambient temperature in RNAlater®. Producer defined 2: B&T Kit @ 4C (RNAlater) Treatment 2: Samples extracted with the Blood and Tissue Kit-based DNA extraction protocol following storage at 4°C in RNAlater®. Producer defined 3: B&T Kit @ 4C Treatment 3: Samples extracted with the Blood and Tissue Kit-based DNA extraction protocol following storage at 4°C without any preservative. Producer defined 4: B&T Kit @ -20C Treatment 4: Samples extracted with the Blood and Tissue Kit-based DNA extraction protocol following storage at -20°C without any preservative. Producer defined 5: PrepMan @ 4C Treatment 5: Samples extracted with the PrepMan-based DNA extraction protocol following storage at 4°C without any preservative. Producer defined 6: PrepMan @ -20C Treatment 6: Samples extracted with the PrepMan-based DNA extraction protocol following storage at -20°C without any preservative. Producer defined Conidia Quantity of conidia included in inoculate for that sample. Producer Defined 100K 100,000 conidia Producer defined 10K 10,000 conidia Producer defined 1K 1,000 conidia Producer defined Time Duration in which samples were stored at respective treatment conditions after inoculation and prior to DNA extraction and qPCR. Producer Defined 1 Month 1 Month Producer defined 3 Months 3 Months Producer defined 3 Days* 3 Days*: Asterisk (*) indicates that the samples from this data set were inoculated with a separate batch of Pd conidia than those prepared for the 1 Month and 3 Months durations. Producer defined Copies \Copies of target DNA detected from qPCR based on a conversion of the Ct value (Cq value) using the standard curve equation associated with each PCR plate. The values were produced from the qPCR machine output. As such, there will be some slight rounding error when converting the reported Ct values with the reported standard curve equations. Producer Defined << empty cell >> Samples with blank cells (n = 6) represent non-detects. Producer defined 0.8188392 69944.9715176 number of copies Ct.value Cycle threshold value, alternatively described as the quantification cycle value (Cq value), produced during qPCR Producer Defined << empty cell >> Samples with blank cells (n = 6) represent non-detects. Producer defined 22.75 39.88 cycles yint Y-intercept value of standard curve equation for respective qPCR plate. Producer Defined 38.71503 39.89476 cycles eff Efficiency of standard curve for respective qPCR plate. Producer Defined 0.908521368612663 0.982991450994503 slope Slope of standard curve equation for respective qPCR plate. Producer Defined -3.56256 -3.36337 r2 R-squared value (coefficient of determination) for standard curve equation of respective qPCR plate. Producer Defined 0.9985 0.9999 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/P13RB3ZK None 20250512 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