http://lod.bco-dmo.org/id/dataset/821955 eng; USA utf8 dataset Highest level of data collection, from a common set of sensors or instrumentation, usually within the same research project Biological and Chemical Oceanography Data Management Office (BCO-DMO) Unavailable 508-289-2009 WHOI MS#36 Woods Hole MA 02543 USA info@bco-dmo.org http://www.bco-dmo.org Monday - Friday 8:00am - 5:00pm For questions regarding this resource, please contact BCO-DMO via the email address provided. pointOfContact 2020-08-25 ISO 19115-2 Geographic Information - Metadata - Part 2: Extensions for Imagery and Gridded Data ISO 19115-2:2009(E) Gene copy number counts and 16S analyses of July 2019 Rhode River surface water incubated with or without viral dilution 2020-08-25 publication 2020-08-25 revision Marine Biological Laboratory/Woods Hole Oceanographic Institution Library (MBLWHOI DLA) 2020-12-07 publication https://doi.org/10.26008/1912/bco-dmo.821955.1 Sarah Preheim Johns Hopkins University principalInvestigator Biological and Chemical Oceanography Data Management Office (BCO-DMO) Unavailable 508-289-2009 WHOI MS#36 Woods Hole MA 02543 USA info@bco-dmo.org http://www.bco-dmo.org Monday - Friday 8:00am - 5:00pm For questions regarding this resource, please contact BCO-DMO via the email address provided. publisher Cite this dataset as: Preheim, S. (2020) Gene copy number counts and 16S analyses of July 2019 Rhode River surface water incubated with or without viral dilution. Biological and Chemical Oceanography Data Management Office (BCO-DMO). (Version 1) Version Date 2020-08-25 [if applicable, indicate subset used]. doi:10.26008/1912/bco-dmo.821955.1 [access date] Gene copy number counts and 16S analyses of July 2019 Rhode River surface water incubated with or without viral dilution Dataset Description: &lt;p&gt;Gene copy number counts and 16S analyses of July 2019 Rhode River surface water incubated with or without viral dilution.&lt;/p&gt; Methods and Sampling: &lt;p&gt;Surface water samples were collected for subsequent microbial community analyses via PCR and shotgun metagenomics. A YSI EXO2 water quality sonde operated by the Smithsonian Environmental Research Center was used to collect physicochemical conditions at time of sample collection.&lt;/p&gt; &lt;p&gt;Shotgun metagenomic DNA and 16S rRNA microbial genes were amplified as described in Sakowski et al. (in revision). Metagenomic DNA was prepped using the Nextera Flex library prep kit. 16S rRNA libraries were prepped by V3-V4 PCR amplification. Both shotgun metagenome and 16S libraries were sequenced on an Illumina MiSeq.&lt;/p&gt; &lt;p&gt;&lt;strong&gt;qPCR of 16S rRNA genes and Viral Ribonucleotide Reductase Genes:&lt;/strong&gt;&lt;br /&gt; Bacterial and viral abundances were estimated by quantitative PCR. For Ribonucleotide Reductase (RNR) quantification, an environmental RNR amplicon was first cloned into chemically competent&amp;nbsp;&lt;em&gt;Escherichia coli&amp;nbsp;&lt;/em&gt;cells using the Zero Blunt PCR Cloning Kit (Thermo Scientific) following the manufacturer's protocol. Environmental samples were quantitated for 16S rRNA and RNR gene copy numbers by comparing to a serial dilution of the cloned&amp;nbsp;&lt;em&gt;E. coli&amp;nbsp;&lt;/em&gt;cells at known concentrations. All standards and environmental samples were run in triplicate. Three microliters of sample were combined with UltraPure molecular grade water (Thermo, Inc.), SsoAdvanced Universal SYBR Green Supermix (1x final concentration, Bio-Rad Laboratories, Inc.), Forward primer (0.3 mM final concentration), and Reverse primer (0.3 mM final concentration) to a final volume of 25 mL. Samples were amplified on a CFX96 Real-Time PCR Detection System (Bio-Rad Laboratories, Inc.) with the following conditions: denaturing at 98°C for 10 minutes; 45 cycles of denaturing at 98°C for 10 seconds, annealing at 52°C for 30 seconds, and extension at 72°C for 45 seconds; and a final extension of 72°C for 5 minutes.&lt;/p&gt; &lt;p&gt;&lt;strong&gt;16S rRNA amplification and sequencing:&lt;/strong&gt;&lt;br /&gt; 16S rRNA genes were amplified from surface water samples in a 25 mL PCR reaction with the following conditions: three microliters of column-purified DNA were combined with UltraPure molecular grade water (Thermo, Inc.), 10X buffer (1x final concentration), dNTPs (0.1mM each final concentration), 16S forward primer 27F (0.3 mM final concentration), 16S reverse primer PE_16S_V4_E786_R (0.3 mM final concentration), bovine serum albumin (0.02 mg/mL final concentration), and Phusion High-Fidelity DNA Polymerase (0.5U; New England BioLabs, Inc. PCR reactions were combined with 150 mL of 4% UMIL EM90 oil (4% UMIL EM90 oil, 0.05% TritonX-100 v/v in mineral oil; Universal Preserv-A-Chem, Inc.) and emulsified by vortexing at max speed (~2,700 rpm) for one minute on a Vortex Genie 2 (MoBio). Emulsions were loaded as 50 mL aliquots and amplified with the following conditions: denaturation at 94°C for 3 minutes; 33 cycles of denaturation at 94°C for 10 seconds, annealing at 54°C for 30 seconds, and extension at 72°C for 45 seconds; and a final extension of 72°C for 5 minutes (C1000, BioRad Labs., Inc.).&lt;/p&gt; &lt;p&gt;PCR oil emulsions were broken with isobutanol. Briefly, PCR aliquots were pooled in a 1.5mL microcentrifuge tube and combined with 100 uL of sterile 5M NaCl solution and 1 mL of isobutanol. Samples were vortexed briefly to mix and centrifuged at 16,000 x g for 1 minute. The bottom aqueous layer was retained, and DNA was purified by spin column purification (Zymo, Inc.). DNA was eluted in 20 uL of Tris-HCl.&lt;/p&gt; &lt;p&gt;Purified DNA was run on a 1.5% agarose gel (UltraPure Agarose, ThermoFisher Scientific). 16S rRNA gene bands were visualized under blue light excitation, extracted, and gel purified (Zymo, Inc.) Purified DNA was eluted into 20 mL of Tris-HCl. Barcodes and Illumina adapters were added to 16S rRNA gene amplicon products in two subsequent limited PCR steps. Barcodes were added as follows: two microliters of purified DNA were combined with UltraPure molecular grade water (Thermo, Inc.), 10X buffer (1x final concentration), dNTPs (0.1mM each final concentration), 16S forward primer PE_16S_V4_U515F (0.3 mM final concentration), 16S rRNA gene reverse primer with 8-mer barcodes PE_IV_XXX (0.3 mM final concentration), and Phusion High-Fidelity DNA Polymerase (0.5U; New England BioLabs, Inc.). Samples were amplified with the following conditions: denaturing at 98°C for 30 seconds; 8 cycles of denaturing at 98°C for 10 seconds, annealing at 54°C for 30 seconds, and extension at 72°C for 45 seconds; and a final extension of 72°C for 5 minutes. DNA was purified by spin column purification (Zymo, Inc.) and eluted into 20 mL Tris-HCl. Illumina adapters were then added as above with the following primers: Illumina adapter forward primer PE-III-PCR-F (0.3 mM final concentration) and Illumina adapter reverse primer Barcode_Rev (0.3 mM final concentration). Samples were amplified with the following conditions: denaturing at 98°C for 30 seconds; 5 cycles of denaturing at 98°C for 10 seconds, annealing at 54°C for 30 seconds, and extension at 72°C for 45 seconds; and a final extension of 72°C for 5 minutes. DNA was purified by spin column purification (Zymo, Inc.) and eluted into 20 mL Tris-HCl. 16S rRNA gene amplicon libraries were sequenced on an Illumina MiSeq (2 x 300 bp) at the Genetic Core Research Facility at Johns Hopkins University.&lt;/p&gt; &lt;p&gt;&lt;strong&gt;Shotgun metagenomic library preparation and sequencing:&lt;/strong&gt;&lt;br /&gt; Surface water samples were filtered through a 0.2µm PES filter. DNA was extracted from filters for shotgun sequencing from water samples with the DNeasy PowerWater kit (Qiagen) following the manufacturer’s protocol with the following amendment: 20 µL of proteinase K was combined with 1 mL of solution PW1 in the bead tube. The bead tube was incubated at 65°C for ten minutes prior to bead beating. Libraries were prepared with the Nextera DNA Flex Library Prep kit (Illumina, Inc.) following the manufacture's protocol and sequenced on an Illumina MiSeq (2 x 300 bp) at the Genetic Core Research Facility at Johns Hopkins University.&lt;/p&gt; &lt;p&gt;&lt;strong&gt;epicPCR of environmental samples:&lt;/strong&gt;&lt;br /&gt; Surface water glycerol samples (25% v/v) were thawed on ice and one mL was added to three replicate 1.5 mL microcentrifuge tubes per sample. Samples were centrifuged at 25,000 x g for 10 minutes and resuspended after supernatant removal to reduce free viral particles. Thirty microliters of each sample was combined with UltraPure molecular grade water (Thermo, Inc.), 10X buffer (1x final concentration), dNTPs (0.1mM each final concentration), Viral Forward primer (1.0 mM final concentration), Viral Reverse_519R fusion primers (R1 and R2 combined, 0.01 mM each final concentration), 16S Reverseeverse primer (1.0 mM final concentration), bovine serum albumin (0.02 mg/mL final concentration), Tween-20 (0.8% v/v final concentration), and Phusion High-Fidelity DNA Polymerase (1.5U; New England BioLabs, Inc.) to a final volume of 75 mL. PCR reactions were combined with 450 mL of 4% UMIL EM90 oil (4% UMIL EM90 oil, 0.05% TritonX-100 v/v in mineral oil; Universal Preserv-A-Chem, Inc.) and emulsified by vortexing at max speed (~2,700 rpm) for one minute. Emulsions were loaded as 50 mL aliquots and amplified with the following conditions: denaturation at 94°C for 3 minutes; 33 cycles of denaturation at 94°C for 10 seconds, annealing at 54°C for 30 seconds, and extension at 72°C for 45 seconds; and a final extension of 72°C for 5 minutes (C1000, BioRad Labs., Inc.).&lt;/p&gt; &lt;p&gt;PCR oil emulsions were broken with isobutanol. Briefly, PCR aliquots were pooled in a 1.5mL microcentrifuge tube and combined with 100 uL of sterile 5M NaCl solution and 1 mL of isobutanol. Samples were vortexed briefly to mix and centrifuged at 16,000 x g for 1 minute. The bottom aqueous layer was retained, and DNA was purified by spin column purification (Zymo, Inc.). DNA was eluted in 20 uL of Tris-HCl.&lt;/p&gt; &lt;p&gt;Amplicons were enriched by nested PCR and subsequently sequenced on a PacBio Sequel with Sequel v3 chemistry (University of Maryland Institute for Genome Sciences). Circular consensus sequences were obtained from raw reads with the following parameters: minimum signal-to-noise ratio (SNR): 3, minimum length: 500bp, minimum passes: 10, minimum read score: 0.75, minimum predicted accuracy: 0.90.&lt;/p&gt; Funding provided by NSF Division of Ocean Sciences (NSF OCE) Award Number: OCE-1820652 Award URL: https://www.nsf.gov/awardsearch/show-award?AWD_ID=1820652 completed Sarah Preheim Johns Hopkins University 410-516-6632 Ames Hall 205 3400 N. Charles St. Baltimore MD 21218 USA spreheim@jhu.edu pointOfContact asNeeded Dataset Version: 1 Unknown Incubation_Time Date Time Temp Conductivity DO_pcnt Pressure pH Chlorophyll_RFU BGA_PE_RFU FDOM_RFU Specific_Conductivity Salinity DO Depth Turbidity Chlorophyll BGA_PE FDOM Location Lat Lon Instrumentation Sample_ID Genes_16S RNR_Genes Library_Accession_Number_16S Illumina MiSeq CFX96 Real-Time PCR Detection System YSI EXO2 water quality sonde theme None, User defined time_elapsed date time_local water temperature at measurement depth conductivity O2 saturation water pressure pH chlorophyll a No BCO-DMO term fluorescence specific conductance salinity dissolved Oxygen depth turbidity site latitude longitude instrument sample identification genetic material concentration accession number featureType BCO-DMO Standard Parameters Automated DNA Sequencer qPCR Thermal Cycler YSI Professional Plus Multi-Parameter Probe instrument BCO-DMO Standard Instruments otherRestrictions otherRestrictions Access Constraints: none. Use Constraints: Please follow guidelines at: http://www.bco-dmo.org/terms-use Distribution liability: Under no circumstances shall BCO-DMO be liable for any direct, incidental, special, consequential, indirect, or punitive damages that result from the use of, or the inability to use, the materials in this data submission. If you are dissatisfied with any materials in this data submission your sole and exclusive remedy is to discontinue use. EAGER: High-throughput, culture-independent technique identifying cyanobacteria infections to improve understanding of carbon biogeochemical cycling https://osprey.bco-dmo.org/project/755237 EAGER: High-throughput, culture-independent technique identifying cyanobacteria infections to improve understanding of carbon biogeochemical cycling <p>NSF Abstract:</p> <p>Viruses in the ocean are 10 times more abundant than bacteria and kill 10-66% of bacterial cells daily. Viral infections of bacteria, such as of photosynthetic cyanobacteria that form the base of the food web, impact the flow of energy and carbon within the marine ecosystem. Thus, viral infections dramatically alter important biogeochemical and ecological factors in the ocean, such as how much carbon dioxide is respired or how many fish the ocean ecosystem can sustain. Despite their importance in the ocean ecosystem, researchers do not know the answer to the most basic question of viral biology for most environmental viruses: which bacteria do different viruses infect? Identifying these infections could help researchers understand more about how viruses shape the ecosystem through infections of keystone microbial species, infections of microbes with unique characteristics, or infection patterns that promote microbial community stability. This project is to develop a cost-effective method to substantially increase the number of infections identified within natural microbial communities. The researchers are applying this novel method to determine viral infections in cyanobacteria in the Chesapeake Bay and compare the results to standard approaches to determine viral infections. This technique can be widely used to transform our understanding of how viruses impact many ecosystems, since it is cost-effective, does not need specialized equipment and can be adapted to target different viral populations. Additionally, this project provides research opportunities for undergraduate and high school students, including underrepresented minority students and women.</p> <p>To develop a high-throughput, culture-independent technique to identify infections in the environment, the researchers are optimizing a previously developed method, emulsion paired concatenation-isolation PCR (epicPCR). Adapting epicPCR for viral-host associations will identify interactions by isolating actively infected single cells within a microdroplet to retain the physical proximity of the host and viral DNA during DNA extraction. Next, fusion PCR is done within the microdroplet to allow host rRNA genes to fuse to viral marker genes, such as g20 or ribonucleotide reductase, retained within the same bead. Only rRNA genes successfully fused to viral markers are amplified. Finally, high-throughput sequencing is done on the resulting fusion products. This approach is cultivation-independent, screens a larger fraction of diversity within the sample than traditional approaches, requires little additional equipment compared to microfluidic approaches, and can be scaled up to hundreds of samples because the amount of sequencing required to deeply sample a single environment is low compared to shotgun metagenomic sequencing. Although this technique will be limited to viral marker genes and suffers from the biases of PCR, it still offers great potential to investigate viral-host interactions across a large number of environments. The method is being applied to determine how viral infections influence cynaobacterial blooms in the Chesapeake Bay. The researchers will also compare the results of epicPCR to culture-based, single-cell, and bioinformatics based methods of host-virus associations to identify biases, limitations and caveats of various approaches.</p> <p>This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.</p> identify cyano infections largerWorkCitation project eng; USA oceans -76.5402 -76.5402 38.8869 38.8869 2019-07-25 2019-07-28 Rhode River waters 38.8869 N, 76.5402 W 0 BCO-DMO catalogue of parameters from Gene copy number counts and 16S analyses of July 2019 Rhode River surface water incubated with or without viral dilution Biological and Chemical Oceanography Data Management Office (BCO-DMO) Unavailable 508-289-2009 WHOI MS#36 Woods Hole MA 02543 USA info@bco-dmo.org http://www.bco-dmo.org Monday - Friday 8:00am - 5:00pm For questions regarding this resource, please contact BCO-DMO via the email address provided. pointOfContact http://lod.bco-dmo.org/id/dataset-parameter/822006.rdf Name: Incubation_Time Units: Hours Description: <p>Time post-incubation</p> http://lod.bco-dmo.org/id/dataset-parameter/822007.rdf Name: Date Units: unitless Description: <p>Sample date; format: YYYY-MM-DD</p> http://lod.bco-dmo.org/id/dataset-parameter/822008.rdf Name: Time Units: unitless Description: <p>Sample Time (EST); format: hh:mm</p> http://lod.bco-dmo.org/id/dataset-parameter/822009.rdf Name: Temp Units: degrees Celsius Description: <p>Water Temperature</p> http://lod.bco-dmo.org/id/dataset-parameter/822010.rdf Name: Conductivity Units: millisiemens per centimeter (mS/cm) Description: <p>Conductivity</p> http://lod.bco-dmo.org/id/dataset-parameter/822011.rdf Name: DO_pcnt Units: % air saturation Description: <p>Water Dissolved Oxygen % air saturation</p> http://lod.bco-dmo.org/id/dataset-parameter/822012.rdf Name: Pressure Units: psi Description: <p>Water pressure</p> http://lod.bco-dmo.org/id/dataset-parameter/822013.rdf Name: pH Units: pH Description: <p>Water pH</p> http://lod.bco-dmo.org/id/dataset-parameter/822014.rdf Name: Chlorophyll_RFU Units: RFU Description: <p>Water Chlorophyll</p> http://lod.bco-dmo.org/id/dataset-parameter/822015.rdf Name: BGA_PE_RFU Units: RFU Description: <p>Water Total Algae</p> http://lod.bco-dmo.org/id/dataset-parameter/822016.rdf Name: FDOM_RFU Units: RFU Description: <p>Fluorescent Dissolved Organic Matter</p> http://lod.bco-dmo.org/id/dataset-parameter/822017.rdf Name: Specific_Conductivity Units: millisiemens per centimeter (mS/cm) Description: <p>Water Specific Conductivity</p> http://lod.bco-dmo.org/id/dataset-parameter/822018.rdf Name: Salinity Units: psu Description: <p>Water Salinity</p> http://lod.bco-dmo.org/id/dataset-parameter/822019.rdf Name: DO Units: milligrams per liter (mg/L) Description: <p>Water Dissolved Oxygen</p> http://lod.bco-dmo.org/id/dataset-parameter/822020.rdf Name: Depth Units: meters Description: <p>Probe Depth</p> http://lod.bco-dmo.org/id/dataset-parameter/822021.rdf Name: Turbidity Units: FNU Description: <p>Water Turbidity</p> http://lod.bco-dmo.org/id/dataset-parameter/822022.rdf Name: Chlorophyll Units: micrograms per liter (ug/L) Description: <p>Water Chlorophyll</p> http://lod.bco-dmo.org/id/dataset-parameter/822023.rdf Name: BGA_PE Units: micrograms per liter (ug/L) Description: <p>Water Total Algae</p> http://lod.bco-dmo.org/id/dataset-parameter/822024.rdf Name: FDOM Units: ppb QSE Description: <p>Fluorescent Dissolved Organic Matter</p> http://lod.bco-dmo.org/id/dataset-parameter/822025.rdf Name: Location Units: SERC Dock Description: <p>Sample Site</p> http://lod.bco-dmo.org/id/dataset-parameter/822026.rdf Name: Lat Units: decimal degrees North Description: <p>Latitude</p> http://lod.bco-dmo.org/id/dataset-parameter/822027.rdf Name: Lon Units: decimal degrees East Description: <p>Longitude</p> http://lod.bco-dmo.org/id/dataset-parameter/822028.rdf Name: Instrumentation Units: unitless Description: <p>Sensor Used for Measurements</p> http://lod.bco-dmo.org/id/dataset-parameter/822029.rdf Name: Sample_ID Units: unitless Description: <p>Unique Sample Identifier</p> http://lod.bco-dmo.org/id/dataset-parameter/822030.rdf Name: Genes_16S Units: gene copy number mean per mL Description: <p>16S qPCR Gene Copy Number Mean</p> http://lod.bco-dmo.org/id/dataset-parameter/822031.rdf Name: RNR_Genes Units: gene copy number mean per mL Description: <p>RNR qPCR Gene Copy Number Mean</p> http://lod.bco-dmo.org/id/dataset-parameter/822032.rdf Name: Library_Accession_Number_16S Units: unitless Description: <p>NCBI Accession Numbers for 16S Libraries</p> GB/NERC/BODC > British Oceanographic Data Centre, Natural Environment Research Council, United Kingdom Biological and Chemical Oceanography Data Management Office (BCO-DMO) Unavailable 508-289-2009 WHOI MS#36 Woods Hole MA 02543 USA info@bco-dmo.org http://www.bco-dmo.org Monday - Friday 8:00am - 5:00pm For questions regarding this resource, please contact BCO-DMO via the email address provided. pointOfContact 3030 https://darchive.mblwhoilibrary.org/bitstream/1912/26446/1/dataset-821955_serc-2019-viral-dilution-incubations__v1.tsv download https://doi.org/10.26008/1912/bco-dmo.821955.1 download onLine dataset &lt;p&gt;Surface water samples were collected for subsequent microbial community analyses via PCR and shotgun metagenomics. A YSI EXO2 water quality sonde operated by the Smithsonian Environmental Research Center was used to collect physicochemical conditions at time of sample collection.&lt;/p&gt; &lt;p&gt;Shotgun metagenomic DNA and 16S rRNA microbial genes were amplified as described in Sakowski et al. (in revision). Metagenomic DNA was prepped using the Nextera Flex library prep kit. 16S rRNA libraries were prepped by V3-V4 PCR amplification. Both shotgun metagenome and 16S libraries were sequenced on an Illumina MiSeq.&lt;/p&gt; &lt;p&gt;&lt;strong&gt;qPCR of 16S rRNA genes and Viral Ribonucleotide Reductase Genes:&lt;/strong&gt;&lt;br /&gt; Bacterial and viral abundances were estimated by quantitative PCR. For Ribonucleotide Reductase (RNR) quantification, an environmental RNR amplicon was first cloned into chemically competent&amp;nbsp;&lt;em&gt;Escherichia coli&amp;nbsp;&lt;/em&gt;cells using the Zero Blunt PCR Cloning Kit (Thermo Scientific) following the manufacturer's protocol. Environmental samples were quantitated for 16S rRNA and RNR gene copy numbers by comparing to a serial dilution of the cloned&amp;nbsp;&lt;em&gt;E. coli&amp;nbsp;&lt;/em&gt;cells at known concentrations. All standards and environmental samples were run in triplicate. Three microliters of sample were combined with UltraPure molecular grade water (Thermo, Inc.), SsoAdvanced Universal SYBR Green Supermix (1x final concentration, Bio-Rad Laboratories, Inc.), Forward primer (0.3 mM final concentration), and Reverse primer (0.3 mM final concentration) to a final volume of 25 mL. Samples were amplified on a CFX96 Real-Time PCR Detection System (Bio-Rad Laboratories, Inc.) with the following conditions: denaturing at 98°C for 10 minutes; 45 cycles of denaturing at 98°C for 10 seconds, annealing at 52°C for 30 seconds, and extension at 72°C for 45 seconds; and a final extension of 72°C for 5 minutes.&lt;/p&gt; &lt;p&gt;&lt;strong&gt;16S rRNA amplification and sequencing:&lt;/strong&gt;&lt;br /&gt; 16S rRNA genes were amplified from surface water samples in a 25 mL PCR reaction with the following conditions: three microliters of column-purified DNA were combined with UltraPure molecular grade water (Thermo, Inc.), 10X buffer (1x final concentration), dNTPs (0.1mM each final concentration), 16S forward primer 27F (0.3 mM final concentration), 16S reverse primer PE_16S_V4_E786_R (0.3 mM final concentration), bovine serum albumin (0.02 mg/mL final concentration), and Phusion High-Fidelity DNA Polymerase (0.5U; New England BioLabs, Inc. PCR reactions were combined with 150 mL of 4% UMIL EM90 oil (4% UMIL EM90 oil, 0.05% TritonX-100 v/v in mineral oil; Universal Preserv-A-Chem, Inc.) and emulsified by vortexing at max speed (~2,700 rpm) for one minute on a Vortex Genie 2 (MoBio). Emulsions were loaded as 50 mL aliquots and amplified with the following conditions: denaturation at 94°C for 3 minutes; 33 cycles of denaturation at 94°C for 10 seconds, annealing at 54°C for 30 seconds, and extension at 72°C for 45 seconds; and a final extension of 72°C for 5 minutes (C1000, BioRad Labs., Inc.).&lt;/p&gt; &lt;p&gt;PCR oil emulsions were broken with isobutanol. Briefly, PCR aliquots were pooled in a 1.5mL microcentrifuge tube and combined with 100 uL of sterile 5M NaCl solution and 1 mL of isobutanol. Samples were vortexed briefly to mix and centrifuged at 16,000 x g for 1 minute. The bottom aqueous layer was retained, and DNA was purified by spin column purification (Zymo, Inc.). DNA was eluted in 20 uL of Tris-HCl.&lt;/p&gt; &lt;p&gt;Purified DNA was run on a 1.5% agarose gel (UltraPure Agarose, ThermoFisher Scientific). 16S rRNA gene bands were visualized under blue light excitation, extracted, and gel purified (Zymo, Inc.) Purified DNA was eluted into 20 mL of Tris-HCl. Barcodes and Illumina adapters were added to 16S rRNA gene amplicon products in two subsequent limited PCR steps. Barcodes were added as follows: two microliters of purified DNA were combined with UltraPure molecular grade water (Thermo, Inc.), 10X buffer (1x final concentration), dNTPs (0.1mM each final concentration), 16S forward primer PE_16S_V4_U515F (0.3 mM final concentration), 16S rRNA gene reverse primer with 8-mer barcodes PE_IV_XXX (0.3 mM final concentration), and Phusion High-Fidelity DNA Polymerase (0.5U; New England BioLabs, Inc.). Samples were amplified with the following conditions: denaturing at 98°C for 30 seconds; 8 cycles of denaturing at 98°C for 10 seconds, annealing at 54°C for 30 seconds, and extension at 72°C for 45 seconds; and a final extension of 72°C for 5 minutes. DNA was purified by spin column purification (Zymo, Inc.) and eluted into 20 mL Tris-HCl. Illumina adapters were then added as above with the following primers: Illumina adapter forward primer PE-III-PCR-F (0.3 mM final concentration) and Illumina adapter reverse primer Barcode_Rev (0.3 mM final concentration). Samples were amplified with the following conditions: denaturing at 98°C for 30 seconds; 5 cycles of denaturing at 98°C for 10 seconds, annealing at 54°C for 30 seconds, and extension at 72°C for 45 seconds; and a final extension of 72°C for 5 minutes. DNA was purified by spin column purification (Zymo, Inc.) and eluted into 20 mL Tris-HCl. 16S rRNA gene amplicon libraries were sequenced on an Illumina MiSeq (2 x 300 bp) at the Genetic Core Research Facility at Johns Hopkins University.&lt;/p&gt; &lt;p&gt;&lt;strong&gt;Shotgun metagenomic library preparation and sequencing:&lt;/strong&gt;&lt;br /&gt; Surface water samples were filtered through a 0.2µm PES filter. DNA was extracted from filters for shotgun sequencing from water samples with the DNeasy PowerWater kit (Qiagen) following the manufacturer’s protocol with the following amendment: 20 µL of proteinase K was combined with 1 mL of solution PW1 in the bead tube. The bead tube was incubated at 65°C for ten minutes prior to bead beating. Libraries were prepared with the Nextera DNA Flex Library Prep kit (Illumina, Inc.) following the manufacture's protocol and sequenced on an Illumina MiSeq (2 x 300 bp) at the Genetic Core Research Facility at Johns Hopkins University.&lt;/p&gt; &lt;p&gt;&lt;strong&gt;epicPCR of environmental samples:&lt;/strong&gt;&lt;br /&gt; Surface water glycerol samples (25% v/v) were thawed on ice and one mL was added to three replicate 1.5 mL microcentrifuge tubes per sample. Samples were centrifuged at 25,000 x g for 10 minutes and resuspended after supernatant removal to reduce free viral particles. Thirty microliters of each sample was combined with UltraPure molecular grade water (Thermo, Inc.), 10X buffer (1x final concentration), dNTPs (0.1mM each final concentration), Viral Forward primer (1.0 mM final concentration), Viral Reverse_519R fusion primers (R1 and R2 combined, 0.01 mM each final concentration), 16S Reverseeverse primer (1.0 mM final concentration), bovine serum albumin (0.02 mg/mL final concentration), Tween-20 (0.8% v/v final concentration), and Phusion High-Fidelity DNA Polymerase (1.5U; New England BioLabs, Inc.) to a final volume of 75 mL. PCR reactions were combined with 450 mL of 4% UMIL EM90 oil (4% UMIL EM90 oil, 0.05% TritonX-100 v/v in mineral oil; Universal Preserv-A-Chem, Inc.) and emulsified by vortexing at max speed (~2,700 rpm) for one minute. Emulsions were loaded as 50 mL aliquots and amplified with the following conditions: denaturation at 94°C for 3 minutes; 33 cycles of denaturation at 94°C for 10 seconds, annealing at 54°C for 30 seconds, and extension at 72°C for 45 seconds; and a final extension of 72°C for 5 minutes (C1000, BioRad Labs., Inc.).&lt;/p&gt; &lt;p&gt;PCR oil emulsions were broken with isobutanol. Briefly, PCR aliquots were pooled in a 1.5mL microcentrifuge tube and combined with 100 uL of sterile 5M NaCl solution and 1 mL of isobutanol. Samples were vortexed briefly to mix and centrifuged at 16,000 x g for 1 minute. The bottom aqueous layer was retained, and DNA was purified by spin column purification (Zymo, Inc.). DNA was eluted in 20 uL of Tris-HCl.&lt;/p&gt; &lt;p&gt;Amplicons were enriched by nested PCR and subsequently sequenced on a PacBio Sequel with Sequel v3 chemistry (University of Maryland Institute for Genome Sciences). Circular consensus sequences were obtained from raw reads with the following parameters: minimum signal-to-noise ratio (SNR): 3, minimum length: 500bp, minimum passes: 10, minimum read score: 0.75, minimum predicted accuracy: 0.90.&lt;/p&gt; Specified by the Principal Investigator(s) &lt;p&gt;Data Processing:&lt;br /&gt; Sequence data were analyzed using the QIIME2 pipeline.&amp;nbsp;&lt;/p&gt; &lt;p&gt;BCO-DMO Processing Notes:&lt;br /&gt; - Split the latitude and longitude values into independent columns;&lt;br /&gt; - converted longitude from degrees West to degrees East;&lt;br /&gt; -&amp;nbsp;modified parameter names to conform with BCO-DMO naming conventions;&lt;br /&gt; -&amp;nbsp;blank values replaced with no data value 'nd'.&lt;/p&gt; Specified by the Principal Investigator(s) asNeeded 7.x-1.1 Biological and Chemical Oceanography Data Management Office (BCO-DMO) Unavailable 508-289-2009 WHOI MS#36 Woods Hole MA 02543 USA info@bco-dmo.org http://www.bco-dmo.org Monday - Friday 8:00am - 5:00pm For questions regarding this resource, please contact BCO-DMO via the email address provided. pointOfContact Illumina MiSeq Illumina MiSeq PI Supplied Instrument Name: Illumina MiSeq Instrument Name: Automated DNA Sequencer Instrument Short Name:Automated Sequencer Instrument Description: A DNA sequencer is an instrument that determines the order of deoxynucleotides in deoxyribonucleic acid sequences. CFX96 Real-Time PCR Detection System CFX96 Real-Time PCR Detection System PI Supplied Instrument Name: CFX96 Real-Time PCR Detection System Instrument Name: qPCR Thermal Cycler Instrument Short Name:qPCR Instrument Description: An instrument for quantitative polymerase chain reaction (qPCR), also known as real-time polymerase chain reaction (Real-Time PCR). YSI EXO2 water quality sonde YSI EXO2 water quality sonde PI Supplied Instrument Name: YSI EXO2 water quality sonde PI Supplied Instrument Description:YSI EXO2 water quality sonde operated by the Smithsonian Environmental Research Center Instrument Name: YSI Professional Plus Multi-Parameter Probe Instrument Short Name:YSI ProPlus Instrument Description: The YSI Professional Plus handheld multiparameter meter provides for the measurement of a variety of combinations for dissolved oxygen, conductivity, specific conductance, salinity, resistivity, total dissolved solids (TDS), pH, ORP, pH/ORP combination, ammonium (ammonia), nitrate, chloride and temperature. More information from the manufacturer.