This Soil Microbiome SFA Viral Production Counts readme.txt file was generated on 2025-10-22 by Amy Zimmerman GENERAL INFORMATION 1. Title of Dataset: Viral Production 16S rRNA Count and Respiration Data 2. Principal Researcher: Name: Kirsten Hofmockel Institution: Pacific Northwest National Laboratory Email: kirsten.hofmockel@pnnl.gov ORCID: 0000-0003-1586-2167 3. Additional Author Contact Information Name: Amy Zimmerman Institution: Pacific Northwest National Laboratory Email: amy.zimmerman@pnnl.gov ORCID: 0000-0001-6709-8274 4. Information about funding sources supporting the data: This program is supported by the U. S. Department of Energy, Office of Science, Office of Biological and Environmental Research, through the Genomic Science Program, under FWP 70880. 5. Geographic location of data collection: 46°15'04"N, 119°43'43"W, Prosser, WA, USA 6. Date of data collection: 2024-06-05 (initial soil collection); 2024-06-11 to 2024-06-13 (collection of incubated soil) DATA & FILE OVERVIEW 1. File List: SoilMicrobiomeSFA_viral-production_qPCR.csv: Sample-specific 16S rRNA gene copy numbers determined by qPCR (proxy of genomic DNA). SoilMicrobiomeSFA_viral-production_counts.csv : Viral and bacterial counts determined by microscopy. SoilMicrobiomeSFA_viral-production_respiration.csv: Whole soil respiration measured periodically as CO2 production over 48 hr incubation. 2. Relationship between files, if important: The three CSV files represent different data streams generated from the same soil incubation experiment, using distinct instrument measurements. Variables “sample_cat”, “treatment”, “time_h”, “rep”, “collection_date” are common variables across all files. 3. Additional related data collected that was not included in the current data package: Associated 16S rRNA amplicon sequence results are included in a separate zip file. 4. Are there multiple versions of the dataset? No DATA-SPECIFIC INFORMATION FOR: SoilMicrobiomeSFA_viral-production_qPCR.csv 1. Number of variables: 7 2. Number of cases/rows: 31, including header row 3. Variable List: sample_cat: unique Soil Microbiome SFA sample catalog number in format “SM####” treatment: viral treatment as “field_abund” or “reduced_abund” to indicate whether viruses were added at measured field abundance (1.6 x109 viruses gdw-1 soil) or at 10% of measured field abundance (1.6 x 108 viruses gdw-1 soil) time_h: numeric hours of incubation rep: numeric condition replicate number (none resampled over time), 1-5 collection_date: date of sample collection (YYYY-MM-DD) date_assay: date qPCR assay was run (YYYY-MM-DD) gene_copies_gdw: Number of 16S rRNA gene copies per gram dry weight soil 4. Missing data codes: NA 5. Specialized formats or other abbreviations used: None DATA-SPECIFIC INFORMATION FOR: SoilMicrobiomeSFA_viral-production_counts.csv 1. Number of variables: 9 2. Number of cases/rows: 71, including header row 3. Variable List: sample_cat: unique Soil Microbiome SFA sample catalog number in format “SM####” treatment: viral treatment as “field_abund” or “reduced_abund” to indicate whether viruses were added at measured field abundance (1.6 x109 viruses gdw-1 soil) or at 10% of measured field abundance (1.6 x 108 viruses gdw-1 soil) time_h: numeric hours of incubation rep: numeric condition replicate number (none resampled over time), 1-5 collection_date: date of sample collection (YYYY-MM-DD) collection_time_hr: time of sample collection (HH:MM) ISO 8601 Bac_gdw: bacterial counts in cells per gram dry weight soil determined by microscopy Viruses_gdw: viral counts in viruses per gram dry weight soil determined by microscopy VBR: virus to bacteria ratio based on Bac_gdw and Viruses_gdw 4. Missing data codes: None 5. Specialized formats or other abbreviations used: gdw == grams dry weight soil DATA-SPECIFIC INFORMATION FOR: SoilMicrobiomeSFA_viral-production_respiration.csv 1. Number of variables: 9 2. Number of cases/rows: 101, including header row 3. Variable List: sample_cat: unique Soil Microbiome SFA sample catalog number in format “SM####” treatment: viral treatment as “field_abund” or “reduced_abund” to indicate whether viruses were added at measured field abundance (1.6 x109 viruses gdw-1 soil) or at 10% of measured field abundance (1.6 x 108 viruses gdw-1 soil) time_h: numeric hours of incubation rep: numeric condition replicate number (none resampled over time), 1-5 collection_date: date of sample collection (YYYY-MM-DD) time_capped: time jar was capped to trap evolved CO2 (HH:MM) time_sampled: time jar headspace was sampled for CO2 concentration (HH:MM) ug_CO2_C_gdw: Total evolved CO2 calculated as the area under the rate-time curve assuming the rate changes linearly between consecutive measurement points, represented as micrograms carbon in CO2 (“CO2-C”) normalized to gram dry weight soil ug_CO2_C_gdw_h: Calculated respiration rate as micrograms carbon in CO2 (“CO2-C”) normalized to gram dry weight soil per hour 4. Missing data codes: None 5. Specialized formats or other abbreviations used: gdw == grams dry weight soil, ug == micrograms METHODOLOGICAL INFORMATION 1. Description of methods used for collection/generation of data: This study aimed to quantify rates of viral production in soil from different viral abundance treatments under conditions as close to natural field soil as possible given the perturbations necessary to manipulate viral abundances. Viruses were removed from soil, then added back to virus-depleted soil to control the initial viral abundances at either 100% (field_abund) or 10% (reduced_abund) of measured field abundance to create treatments with field-relevant or reduced viral infection pressure. Replicates (n=5) of batch incubation jars were harvested every 8 hours for 48 hours to enumerate bacteria and viruses by microscopy and profile bacterial community composition by 16S rRNA amplicon sequencing. To directly count viruses by epifluorescence microscopy, they were extracted from the soil matrix. Aliquots of frozen soil from each harvest time were moved from the -80ºC freezer to a 4ºC refrigerator for one hour prior to extraction to allow soil to gently thaw. Virus extractions were performed according to the Emerson Lab Soil Viromics protocol (https://www.protocols.io/view/soil-viromics-protocol-emerson-lab-v1-kxygxz7q4v8j/v1) with a slight modification to the buffer chemistry. BSA was excluded from the extraction buffer because it interfered with fluorescence staining and microscopy. We added 18 mL 1% potassium citrate in phosphate buffered saline solution (10 g/L potassium citrate, 1.44 g/L Na2HPO4 ⋅ 7H2O, and 0.24 g/L KH2PO4) to each 50 mL tube containing 20 g (wet weight) incubated soil. Samples were shaken horizontally at 300 rpm for 10 minutes at 4ºC. Tubes were then centrifuged in a fixed angle rotor at 4,000 rpm for 10 minutes to pellet the soil. The virus-containing supernatant was decanted into a new 50 mL tube and stored at 4ºC in between steps. Another 18 mL of potassium citrate buffer was added to the soil pellet and vortexed to resuspend the soil for another round of extraction (shaking and centrifugation). The resulting virus-containing supernatant was pooled with the supernatant from the first extraction. A third round of extraction was completed on the same soil, producing approximately 54 mL total of virus-containing supernatant. The pooled viral solution was centrifuged for 8 minutes at 10,000 x g at 4°C in fixed angle rotor to pellet any remaining soil then filtered through 0.22 µm PES (Sartolab RF 50, Sartorius) to exclude bacteria. The filtered viral supernatant was stored at 4ºC for up to one day prior to enumeration by epifluorescence microscopy. Bacteria were extracted from each destructive sampling time point according to Liang et. al., 2020 (https://doi.org/10.3389/fmicb.2020.01287). To enumerate viruses or bacteria in harvested samples, the viral and bacterial extracts were analyzed using epifluorescence microscopy. To reduce background fluorescence, extracts (100 uL) were DNAse treated (Promega MG6101) to digest extracellular DNA following manufacturer instructions. For viruses, the samples were digested at 37ºC for 2.5 hours to ensure complete digestion of extracellular genomic material, while bacterial samples were digested for 30 minutes. Viral and bacterial enumeration differed only in the filters used. DNase-treated viral samples were filtered onto 0.02 µm Anodisc filters (Watman, Cytiva), while bacteria were filtered into 0.2 µm filters. Each filter was stained with 500 uL 2X SybrGold Nucleic Acid Gel Stain (Invitrogen) for 20 minutes in the dark. Filters were mounted onto glass slides with sterile, 0.1 µm filtered 50/50 Glycerol/PBS mounting solution and imaged using a Leica microscope. For each filter, fifteen images were captured to average the number of particles/cells across the whole filter. DNA was extracted from a subset of frozen soil samples for 16S rRNA amplicon sequencing and qPCR analysis. Because all jars were independently sampled (i.e., no jars were re-sampled through time except for respiration measurements), 3 of the 5 jar replicates with the highest viral abundance were chosen from each of 5 time points (0, 16, 24, 32, and 48 hours) for DNA extraction. For each sample, 250 mg of soil was extracted using the Zymo Quick-DNA Fecal/Soil Microbe Miniprep Kit D6010 per the manufacturer’s instructions. DNA was eluted in 50 µL of Zymo elution buffer. DNA yield was quantified by Qubit DNA High Sensitivity assay (Invitrogen) and quality was checked by NanoDrop (Thermo Fisher Scientific) spectrophotometry. Triplicate qPCR reactions were run to quantify genomic DNA as the number of 16SrRNA gene copies per gram dry soil. Each reaction contained 0.25 uM of forward and reverse primers, 1X SsoAdvanced Universal SYBR Green Master Mix (Bio-Rad), and 1.5 mM MgCl2 (10 uL total). Bacterial 16S rRNA gene copies from extracted soil DNA were quantified using primers EUB338 5′-ACT CCT ACG GGA GGC AGC AG-3′ and EUB518 5′-ATT ACC GCG GCT GCT GG-3′. Bacterial 16S quantification assays were run at 98°C for 3 min with 40 cycles of 98°C for 30 sec, 53°C for 45 sec, and 72°C for 30 sec on a CFX384 Touch Real-Time PCR cycler (Bio-Rad). Standard curves were run in triplicate and consisted of a serial dilution of Escherichia coli strain K-12 genomic DNA. A melt curve analysis was constructed by increasing the temperature from 65°C to 95°C. 2. Methods for processing the data: Microscopy images were analyzed using the “analyze particles” command within the software ImageJ (Schneider et al., 2012; https://doi.org/10.1038/nmeth.2089) to obtain counts of cells/particles per frame. The average number of particles per frame was extrapolated to the usable size of the filter surface (i. e. the inner diameter of the filtering funnel applied to the filter surface) to determine the total number of viruses or bacteria on the filter, which is equivalent to the total number of viruses or bacteria per mL of solution added to the filter surface. This concentration was then converted to viruses or cells per gram dry weight soil. 3. Instrument- or software-specific information needed to interpret the data: NA 4. Standards and calibration information, if appropriate: NA 5. Environmental/experimental conditions: Field Collection Soil was collected from the Irrigated Agriculture Research and Extension Center (IAREC) in Prosser, WA (46.251601, -119.728760) from a managed tall wheatgrass (Thinopyrum ponticum) field under drip irrigation applied at 100% of soil field capacity and described previously. Soil was collected three times to prepare for laboratory-based incubation: soil was collected in April and May 2024 to generate viral inoculum; additional soil was collected in June 2024 to generate the virus-depleted soil/bacterial inoculum and dissolved organic matter solution that were used to establish each incubation jar. Viruses extracted from both April and May samples were combined and concentrated prior to inoculation. On each sampling date, >2 kg soil was collected from 0-15 cm in the edges of 100% irrigation plots and transported back to the laboratory in Richland, WA on ice in a cooler and stored at 4°C overnight before processing. Soil was homogenized through 4 mm sieves after removing rocks and roots. Sieved soil was stored at 4°C until further processing (either preparation of viral inoculum or soil inoculum and DOM solution), which was completed within a week of each sample collection. Soil Incubation Design Soil batch incubations included a field equivalent and a reduced viral abundance treatment, where viruses were added back to the soil/bacterial inoculum at either 100% (1.6 × 109 viruses gdw-1 soil; field_abund) or 10% (1.6 × 108 viruses gdw-1 soil; reduced abund) of the original soil virus abundance, respectively. The original virus abundance was determined by epifluorescence microscopy as viruses per gram dry weight (gdw) in collected field soil. All incubations were initiated by adding 65 g air-dried virus-depleted soil/bacterial inoculum to 236 ml wide-mouth Ball mason jars and adding solution to reach a final gravimetric water content of 18%. Soil in each jar was gently mixed with an ethanol-sterilized metal scoopula to distribute moisture evenly. We established 5 replicates of each viral treatment for each sample harvest (0, 8, 16, 24, 32, 40, and 48 hours). Jars were sealed with breathable parafilm to allow gas exchange while excluding potential airborne contaminants and incubated at 25°C. Soil incubations were harvested at seven time points: just after initiation (T0) and after 8, 16, 24, 32, 40, or 48 hours of incubation. At each harvest time, each jar’s soil was mixed with a sterile spatula and subsampled into separate tubes (Olympus Plastics) for viral extraction and enumeration (20g), bacterial extraction and enumeration (10g), and amplicon sequencing (2g). All tubes were immediately flash frozen in liquid nitrogen and stored at -80℃ until further processing. 6. Describe any quality-assurance procedures performed on the data: None 7. People involved with sample collection, processing, analysis and/or submission: Regan McDearis, Sheryl Bell, Sharon Zhao, Evan Warburton, Lupita Renteria, Nicholas Reichart, Amy Zimmerman, Kirsten Hofmockel SHARING/ACCESS INFORMATION 1. Licenses/restrictions placed on the data: This work is marked with CC0 1.0: https://creativecommons.org/publicdomain/zero/1.0/. The authors do request that you appropriately cite the dataset when referencing or re-using the dataset. 2. Links to publications that cite or use the data: NA 3. Links to other publicly accessible locations of the data: NA 4. Links/relationships to ancillary data sets: NA 5. Was data derived from another source? No 6. Recommended citation for this dataset: McDearis, R., A.E. Zimmerman, S.L. Bell, K.S. Hofmockel. 2026. Soil viral production count, respiration, and amplicon data. [Data Set] PNNL DataHub. https://doi.org/10.25584/3653988