http://lod.bco-dmo.org/id/dataset/828392
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
2020-11-05
ISO 19115-2 Geographic Information - Metadata - Part 2: Extensions for Imagery and Gridded Data
ISO 19115-2:2009(E)
High throughput tag-sequencing data from Gorda Ridge Hydrothermal vent field, including 16S and 18S rRNA gene sequences, and environmental metadata from Gorda Ridge Seamount, May/June 2019
2020-11-09
publication
2020-11-09
revision
Marine Biological Laboratory/Woods Hole Oceanographic Institution Library (MBLWHOI DLA)
2021-01-06
publication
https://doi.org/10.26008/1912/bco-dmo.828392.1
Sarah K. Hu
Woods Hole Oceanographic Institution
principalInvestigator
Julie Huber
Woods Hole Oceanographic Institution
principalInvestigator
Amy R. Smith
Woods Hole Oceanographic Institution
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: Hu, S. K., Huber, J., Smith, A. R. (2021) High throughput tag-sequencing data from Gorda Ridge Hydrothermal vent field, including 16S and 18S rRNA gene sequences, and environmental metadata from Gorda Ridge Seamount, May/June 2019. Biological and Chemical Oceanography Data Management Office (BCO-DMO). (Version 1) Version Date 2020-11-09 [if applicable, indicate subset used]. doi:10.26008/1912/bco-dmo.828392.1 [access date]
Gorda Ridge SRA Dataset Description: <p>See SRA BioProject accession: PRJNA637089 (https://www.ncbi.nlm.nih.gov/bioproject/PRJNA637089) and&nbsp;BioSample accession IDs: SAMN15341559-SAMN15341619.&nbsp;Sequences are available&nbsp;via SRA (NCBI).</p>
<p>&nbsp;</p> Methods and Sampling: <p><strong>Sample collection:</strong></p>
<p>The Gorda Ridge spreading center, ~200 km off the coast of southern Oregon was visited in May-June 2019 with the EV Nautilus (cruise NA108). Cruise operations were a collaborative effort with OET, NASA-SUBSEA, NOAA, URI, and WHOI. The ROV Hercules was positioned next to sites visually identified to have active hydrothermal flow. Temperatures at sites of active diffuse flow were obtained using a temperature probe controlled by the ROV Hercules arm. The SUPR sampler intake value then pumped this fluid into air-tight bags for grazing experiments and whole vent fluid water collection or pumped this fluid across 0.2 um PES Millipore filters (size, manufacturer). Fluid was also collected within the plume above each vent site by situating the ROV ~5 m above an active venting site and firing Niskin bottles. Background seawater from the water column at ~2,100 m was obtained by Niskin bottle. Samples from the plume and background collected by emptying Niskin bottles into acid-washed cubitainers and processed in the lab as described below. Upon recovery of ROV Hercules from each dive, bags filled with hydrothermal vent fluid were processed immediately. Filters were placed in RNAlater and kept frozen until return to the lab and fluid collected into bags was processed for grazing experiments (see 18S rRNA gene sequences) or filtered onto sterivex filters (both 18S and 16S sequences).&nbsp;</p>
<p><strong>16S sequences</strong></p>
<p>DNA was extracted from 142 mm filters or Sterivex filters. The 142 mm filters were snap-frozen after folding and then rolling them into 50 mL tubes. To extract DNA, sterile forceps were used to pull the filter out of the tube, ethanol-flamed scissors were used to cut approximately 1/3 of the filter off and into a sterile 15 mL tube, then the rest of the filter was replaced at – 80oC in RNA Later. For the Sterivex filters, the filter housing was cracked open to expose the filter, the filter was cut off with sterile razor blade, and finally placed into a sterile tube with forceps. Approximately ½ of the filter was used, then the rest was saved at - 80oC covered in RNA Later.&nbsp;&nbsp;</p>
<p>All filters were washed in 2 mL 1x sterile PBS by swishing them up and down in the PBS in the 15 mL tubes using sterile forceps. The PBS was transferred to 2 mL tubes and centrifuged at 14,000 rpm for 5 minutes to pellet cells. Filters were then placed in the pellet tubes for digestion. We added 900 uL DNA extraction buffer &nbsp;(50 mL, 100mM Tris, 100mM EDTA, 100mM NaH2PO4, 1.5M NaCl, 1% CTAB), 20 uL proteinase K (10mg/mL), and 40 uL of lysozyme (50mg/mL) to each tube. We proceeded to do freeze-thaw 3 times at -80oC for 15 minutes and thaw at 37oC for 15 minutes. Tubes were then incubated in a 37oC heat block for 20 minutes. Filter-sterile SDS (50 mL of 20% solution in water) was added to teach tube and inverted several times to mix. Tubes were incubated for 2 hours in a 65oC water bath&nbsp;</p>
<p>For the extraction steps, we worked in a fume hood and began by filling each tube to the top with phenol:chloroform:isoamyl alcohol (25:24:1, pH 8.0), and vortexing. Next, we centrifuged at low speed (3000 rpm) for 5 minutes and transferred the top layer to a new 2 mL tube, and repeated (total 2 washes). We added 0.6 volumes of room temperature 100% Isopropanol and inverted gently to mix.&nbsp;Tubes were incubated at room temperature overnight in a dark drawer and then centrifuged at top speed for 30 minutes at 4 οC. We then carefully pipetted off the buffer + isopropanol and saved this fraction in a new 2 mL tube. Ice cold 70% EtOH (1mL) was added to each 2 mL tube, inverted several times, and centrifuged at max speed for 10 min.&nbsp;EtOH was pipetted off and rinsing was repeated two times before allowing the tubes to air dry with a Kim wipe draped over the top. The dried DNA pellet was then dissolved into 50 uL nuclease-free sterile water and quantified using the PicoGreen Assay.&nbsp;</p>
<p>DNA was PCR amplified using the 16S V4 primers 515F (Parada et al. 2016) and 806R (Apprill et al. 2015). Amplicons were sequenced with Illumina MiSeq sequencing.&nbsp;</p>
<p><strong>18S sequences</strong></p>
<p>Samples collected for molecular analyses included in situ filters from the SUPR sampler, shipboard Sterivex filters, or time points from grazing experiments (all collected into 0.2-um pore size filters). Some of the 18S rRNA gene tag sequence samples are associated with grazing experiments, where vent fluid was incubated with analog prey (Fluorescently-labeled bacterial prey) to track grazing rates among single-celled microbial eukaryotes. These samples were taken at the start of the experiments (T0) or at various time points (T1, T2). For all samples, RNA was extracted and amplified similarly to the protocol described in Hu et al. 2018 (https://dx.doi.org/10.17504/protocols.io.hk3b4yn). Frozen filters were thawed and placed into sterile 15-ml falcon tubes with sterile forceps, 1-2 mL of RLT+ buffer (with β-Mercaptoethanol, Qiagen, Valencia, CA, USA), and RNase-free silica beads was added to each tube. Falcon tubes were bead-beaten by vortexing vigorously for 5 minutes. The original sample collection tubes with RNAlater were centrifuged to pellet any cellular material left in the RNAlater; the RNAlater was removed and replaced with 500-ul of RLT+ buffer (with β-Mercaptoethanol). This was vortexed and added to the 15-ml falcon tube. RNA was extracted with the RNAeasy kit (Qiagen #74104) with the in-line genomic DNA removal step (RNase-free DNase reagents, Qiagen #79254). RNA concentrations were determined using the Ribogreen protocol. Extracted RNA was reverse transcribed into cDNA using a cDNA synthesis kit (iScript Select cDNA Synthesis, BioRad, #1708896, Hercules, CA); the concentration of RNA was normalized for the cDNA synthesis reaction (input –ng of RNA). Primers targeting the V4 hypervariable region of the 18S rRNA gene (Stoeck et al. 2010; Hu et al. 2015) were used in PCR reactions, which consisted of a final concentration of 1X Q5 High Fidelity Master Mix (NEB #M0492S, Ipswich, MA), 0.5 μM each of forward and reverse primers, and 1 ng of genetic material. The PCR thermal protocol started with an initial activation step (Q5 specific) of 98°C for 2 min, followed with 10 cycles of 98°C for 10 s, 53°C for 30 s, 72°C for 30 s, and 15 cycles of 98°C for 10 s, 48°C for 30 s, and 72°C for 30 s, and a final extension of 72°C for 2 min (modified from Rodriquez Martinez et al. 2012). The original extract total RNA was also PCR amplified to ensure no genomic DNA was present in the sample. PCR products were checked by confirming the presence of an ~400 bp product on an agarose gel. In cases with no amplification, the PCR reaction was repeated with a higher concentration of cDNA (1.5-2 ng). If this did not yield the expected PCR product, the reaction was repeated with an additional 5 cycles. Three shipboard blanks (MilliQ water) and one extraction blank were also extracted and PCR amplified; while no PCR product was observed in these control samples they were processed similarly to all true samples and sequenced. All PCR products were cleaned using the AMPure bead clean up (Beckman Coulter #A63881, Brea, CA). Samples were multiplexed, pooled at equimolar concentrations and sequenced using the MiSeq 300 x 300 bp PE sequencing at Marine Biological Laboratory Bay Paul Center sequencing facility.&nbsp;</p>
Funding provided by NSF Division of Ocean Sciences (NSF OCE) Award Number: OCE-0939564 Award URL: https://www.nsf.gov/awardsearch/show-award?AWD_ID=0939564
completed
Sarah K. Hu
Woods Hole Oceanographic Institution
skhu@tamu.edu
pointOfContact
Julie Huber
Woods Hole Oceanographic Institution
(508) 289-2556
Woods Hole Oceanographic Institution Marine Chemistry & Geochemistry, Watson Building, MS51
Woods Hole
MA
02543
USA
jhuber@whoi.edu
pointOfContact
Amy R. Smith
Woods Hole Oceanographic Institution
asmith@whoi.edu
pointOfContact
asNeeded
Dataset Version: 1
Unknown
biosample_accession
library_ID
title
library_strategy
library_source
library_selection
library_layout
platform
instrument_model
design_description
filetype
filename_R1
filename_R2
sample_title
bioproject_accession
organism
isolation_source
collection_date
geo_loc_name
lat
lon
samp_collect_device
source_material_id
bacterial_or_eukaryotic_community
Grazing_experiment_or_in_situ
Site_name
Replicate_information
Time_point
sample_id
sample_name
MiSeq 300 x 300 bp PE
Beckman Coulter #A63881, Brea, CA
Niskin bottle
theme
None, User defined
sample identification
sample description
instrument
No BCO-DMO term
file_name
accession number
date
region
latitude
longitude
sampling_method
site
replicate
time_point
featureType
BCO-DMO Standard Parameters
Automated DNA Sequencer
Coulter Counter
Niskin bottle
Thermal Cycler
instrument
BCO-DMO Standard Instruments
NA108
service
Deployment Activity
Gorda Ridges, Pacific Ocean
place
Locations
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.
Center for Dark Energy Biosphere Investigations
http://www.darkenergybiosphere.org
Center for Dark Energy Biosphere Investigations
The mission of the Center for Dark Energy Biosphere Investigations (C-DEBI) is to explore life beneath the seafloor and make transformative discoveries that advance science, benefit society, and inspire people of all ages and origins.
C-DEBI provides a framework for a large, multi-disciplinary group of scientists to pursue fundamental questions about life deep in the sub-surface environment of Earth. The fundamental science questions of C-DEBI involve exploration and discovery, uncovering the processes that constrain the sub-surface biosphere below the oceans, and implications to the Earth system. What type of life exists in this deep biosphere, how much, and how is it distributed and dispersed? What are the physical-chemical conditions that promote or limit life? What are the important oxidation-reduction processes and are they unique or important to humankind? How does this biosphere influence global energy and material cycles, particularly the carbon cycle? Finally, can we discern how such life evolved in geological settings beneath the ocean floor, and how this might relate to ideas about the origin of life on our planet?
C-DEBI's scientific goals are pursued with a combination of approaches:
(1) coordinate, integrate, support, and extend the research associated with four major programs—Juan de Fuca Ridge flank (JdF), South Pacific Gyre (SPG), North Pond (NP), and Dorado Outcrop (DO)—and other field sites;
(2) make substantial investments of resources to support field, laboratory, analytical, and modeling studies of the deep subseafloor ecosystems;
(3) facilitate and encourage synthesis and thematic understanding of submarine microbiological processes, through funding of scientific and technical activities, coordination and hosting of meetings and workshops, and support of (mostly junior) researchers and graduate students; and
(4) entrain, educate, inspire, and mentor an interdisciplinary community of researchers and educators, with an emphasis on undergraduate and graduate students and early-career scientists.
Note: Katrina Edwards was a former PI of C-DEBI; James Cowen is a former co-PI.
Data Management:
C-DEBI is committed to ensuring all the data generated are publically available and deposited in a data repository for long-term storage as stated in their Data Management Plan (PDF) and in compliance with the NSF Ocean Sciences Sample and Data Policy. The data types and products resulting from C-DEBI-supported research include a wide variety of geophysical, geological, geochemical, and biological information, in addition to education and outreach materials, technical documents, and samples. All data and information generated by C-DEBI-supported research projects are required to be made publically available either following publication of research results or within two (2) years of data generation.
To ensure preservation and dissemination of the diverse data-types generated, C-DEBI researchers are working with BCO-DMO Data Managers make data publicly available online. The partnership with BCO-DMO helps ensure that the C-DEBI data are discoverable and available for reuse. Some C-DEBI data is better served by specialized repositories (NCBI's GenBank for sequence data, for example) and, in those cases, BCO-DMO provides dataset documentation (metadata) that includes links to those external repositories.
C-DEBI
largerWorkCitation
program
Probing subseafloor microbial interactions via hydrothermal vent fluids: A focus on protists
https://www.darkenergybiosphere.org/award/probing-subseafloor-microbial-interactions-via-hydrothermal-vent-fluids-a-focus-on-protists/
Probing subseafloor microbial interactions via hydrothermal vent fluids: A focus on protists
<p>Adjusted C-DEBI Award Description:</p>
<p>Highly reduced and thermally charged venting fluids from the subseafloor mix with surrounding seawater, creating a sharp geochemical gradient which promotes a hub of biological diversity at hydrothermal vent ecosystems. While studies of prokaryotic diversity at hydrothermal vent sites have highlighted the important roles microorganisms play in deep sea carbon cycling and offered a unique window into subseafloor microbial communities, depictions of deep-sea marine ecology and food webs are incomplete without characterization of single-celled microbial eukaryotes (protists). I propose to use culture-independent techniques (tag-sequencing and metatranscriptomics) to provide a thorough understanding of protistan biogeography in and near venting fluids, focusing on the vent fluid-seawater interface. Additionally, these qualitative analyses will be paired with quantitative experiments that measure protistan grazing pressure. Understanding trophic interactions within the protistan community is incredibly important, as these processes form the foundation of deep-sea marine food webs and mediate a significant amount of carbon transferred to higher trophic levels.</p>
<p>C-DEBI project link: <a href="https://www.darkenergybiosphere.org/award/probing-subseafloor-microbial-interactions-via-hydrothermal-vent-fluids-a-focus-on-protists/">https://www.darkenergybiosphere.org/award/probing-subseafloor-microbial-interactions-via-hydrothermal-vent-fluids-a-focus-on-protists/ </a></p>
Microbial eukaryotes at hydrothermal vents
largerWorkCitation
project
eng; USA
biota
oceans
Gorda Ridges, Pacific Ocean
-126.743
-126.7048
42.7495
42.7613
2019-05-30
2019-06-07
Axial Seamount Juan de Fuca Ridge NE Pacific 46 N 130 W, Gorda Ridge NE Pacific 41 N 127 W, Mid-Cayman Rise Caribbean Sea 18 N 82 W
0
BCO-DMO catalogue of parameters from High throughput tag-sequencing data from Gorda Ridge Hydrothermal vent field, including 16S and 18S rRNA gene sequences, and environmental metadata from Gorda Ridge Seamount, May/June 2019
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/828584.rdf
Name: biosample_accession
Units: unitless
Description: <p>NCBI BioSample accession number</p>
http://lod.bco-dmo.org/id/dataset-parameter/828585.rdf
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Description: <p>Short unique identifier for the sequencing library.</p>
http://lod.bco-dmo.org/id/dataset-parameter/828586.rdf
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Description: <p>Short description that will identify the dataset on public pages.</p>
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Description: <p>Paired-end or Single</p>
http://lod.bco-dmo.org/id/dataset-parameter/828591.rdf
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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
50026
https://darchive.mblwhoilibrary.org/bitstream/1912/26534/1/dataset-828392_microbial-eukaryotic-diversity-gorda-ridge-sra__v1.tsv
download
https://doi.org/10.26008/1912/bco-dmo.828392.1
download
onLine
dataset
<p><strong>Sample collection:</strong></p>
<p>The Gorda Ridge spreading center, ~200 km off the coast of southern Oregon was visited in May-June 2019 with the EV Nautilus (cruise NA108). Cruise operations were a collaborative effort with OET, NASA-SUBSEA, NOAA, URI, and WHOI. The ROV Hercules was positioned next to sites visually identified to have active hydrothermal flow. Temperatures at sites of active diffuse flow were obtained using a temperature probe controlled by the ROV Hercules arm. The SUPR sampler intake value then pumped this fluid into air-tight bags for grazing experiments and whole vent fluid water collection or pumped this fluid across 0.2 um PES Millipore filters (size, manufacturer). Fluid was also collected within the plume above each vent site by situating the ROV ~5 m above an active venting site and firing Niskin bottles. Background seawater from the water column at ~2,100 m was obtained by Niskin bottle. Samples from the plume and background collected by emptying Niskin bottles into acid-washed cubitainers and processed in the lab as described below. Upon recovery of ROV Hercules from each dive, bags filled with hydrothermal vent fluid were processed immediately. Filters were placed in RNAlater and kept frozen until return to the lab and fluid collected into bags was processed for grazing experiments (see 18S rRNA gene sequences) or filtered onto sterivex filters (both 18S and 16S sequences).&nbsp;</p>
<p><strong>16S sequences</strong></p>
<p>DNA was extracted from 142 mm filters or Sterivex filters. The 142 mm filters were snap-frozen after folding and then rolling them into 50 mL tubes. To extract DNA, sterile forceps were used to pull the filter out of the tube, ethanol-flamed scissors were used to cut approximately 1/3 of the filter off and into a sterile 15 mL tube, then the rest of the filter was replaced at – 80oC in RNA Later. For the Sterivex filters, the filter housing was cracked open to expose the filter, the filter was cut off with sterile razor blade, and finally placed into a sterile tube with forceps. Approximately ½ of the filter was used, then the rest was saved at - 80oC covered in RNA Later.&nbsp;&nbsp;</p>
<p>All filters were washed in 2 mL 1x sterile PBS by swishing them up and down in the PBS in the 15 mL tubes using sterile forceps. The PBS was transferred to 2 mL tubes and centrifuged at 14,000 rpm for 5 minutes to pellet cells. Filters were then placed in the pellet tubes for digestion. We added 900 uL DNA extraction buffer &nbsp;(50 mL, 100mM Tris, 100mM EDTA, 100mM NaH2PO4, 1.5M NaCl, 1% CTAB), 20 uL proteinase K (10mg/mL), and 40 uL of lysozyme (50mg/mL) to each tube. We proceeded to do freeze-thaw 3 times at -80oC for 15 minutes and thaw at 37oC for 15 minutes. Tubes were then incubated in a 37oC heat block for 20 minutes. Filter-sterile SDS (50 mL of 20% solution in water) was added to teach tube and inverted several times to mix. Tubes were incubated for 2 hours in a 65oC water bath&nbsp;</p>
<p>For the extraction steps, we worked in a fume hood and began by filling each tube to the top with phenol:chloroform:isoamyl alcohol (25:24:1, pH 8.0), and vortexing. Next, we centrifuged at low speed (3000 rpm) for 5 minutes and transferred the top layer to a new 2 mL tube, and repeated (total 2 washes). We added 0.6 volumes of room temperature 100% Isopropanol and inverted gently to mix.&nbsp;Tubes were incubated at room temperature overnight in a dark drawer and then centrifuged at top speed for 30 minutes at 4 οC. We then carefully pipetted off the buffer + isopropanol and saved this fraction in a new 2 mL tube. Ice cold 70% EtOH (1mL) was added to each 2 mL tube, inverted several times, and centrifuged at max speed for 10 min.&nbsp;EtOH was pipetted off and rinsing was repeated two times before allowing the tubes to air dry with a Kim wipe draped over the top. The dried DNA pellet was then dissolved into 50 uL nuclease-free sterile water and quantified using the PicoGreen Assay.&nbsp;</p>
<p>DNA was PCR amplified using the 16S V4 primers 515F (Parada et al. 2016) and 806R (Apprill et al. 2015). Amplicons were sequenced with Illumina MiSeq sequencing.&nbsp;</p>
<p><strong>18S sequences</strong></p>
<p>Samples collected for molecular analyses included in situ filters from the SUPR sampler, shipboard Sterivex filters, or time points from grazing experiments (all collected into 0.2-um pore size filters). Some of the 18S rRNA gene tag sequence samples are associated with grazing experiments, where vent fluid was incubated with analog prey (Fluorescently-labeled bacterial prey) to track grazing rates among single-celled microbial eukaryotes. These samples were taken at the start of the experiments (T0) or at various time points (T1, T2). For all samples, RNA was extracted and amplified similarly to the protocol described in Hu et al. 2018 (https://dx.doi.org/10.17504/protocols.io.hk3b4yn). Frozen filters were thawed and placed into sterile 15-ml falcon tubes with sterile forceps, 1-2 mL of RLT+ buffer (with β-Mercaptoethanol, Qiagen, Valencia, CA, USA), and RNase-free silica beads was added to each tube. Falcon tubes were bead-beaten by vortexing vigorously for 5 minutes. The original sample collection tubes with RNAlater were centrifuged to pellet any cellular material left in the RNAlater; the RNAlater was removed and replaced with 500-ul of RLT+ buffer (with β-Mercaptoethanol). This was vortexed and added to the 15-ml falcon tube. RNA was extracted with the RNAeasy kit (Qiagen #74104) with the in-line genomic DNA removal step (RNase-free DNase reagents, Qiagen #79254). RNA concentrations were determined using the Ribogreen protocol. Extracted RNA was reverse transcribed into cDNA using a cDNA synthesis kit (iScript Select cDNA Synthesis, BioRad, #1708896, Hercules, CA); the concentration of RNA was normalized for the cDNA synthesis reaction (input –ng of RNA). Primers targeting the V4 hypervariable region of the 18S rRNA gene (Stoeck et al. 2010; Hu et al. 2015) were used in PCR reactions, which consisted of a final concentration of 1X Q5 High Fidelity Master Mix (NEB #M0492S, Ipswich, MA), 0.5 μM each of forward and reverse primers, and 1 ng of genetic material. The PCR thermal protocol started with an initial activation step (Q5 specific) of 98°C for 2 min, followed with 10 cycles of 98°C for 10 s, 53°C for 30 s, 72°C for 30 s, and 15 cycles of 98°C for 10 s, 48°C for 30 s, and 72°C for 30 s, and a final extension of 72°C for 2 min (modified from Rodriquez Martinez et al. 2012). The original extract total RNA was also PCR amplified to ensure no genomic DNA was present in the sample. PCR products were checked by confirming the presence of an ~400 bp product on an agarose gel. In cases with no amplification, the PCR reaction was repeated with a higher concentration of cDNA (1.5-2 ng). If this did not yield the expected PCR product, the reaction was repeated with an additional 5 cycles. Three shipboard blanks (MilliQ water) and one extraction blank were also extracted and PCR amplified; while no PCR product was observed in these control samples they were processed similarly to all true samples and sequenced. All PCR products were cleaned using the AMPure bead clean up (Beckman Coulter #A63881, Brea, CA). Samples were multiplexed, pooled at equimolar concentrations and sequenced using the MiSeq 300 x 300 bp PE sequencing at Marine Biological Laboratory Bay Paul Center sequencing facility.&nbsp;</p>
Specified by the Principal Investigator(s)
<p><strong>BCO-DMO Processing Notes: </strong><br />
- data submitted in Excel files "Axial-Metagenome.environmental.1.0_JH.xlsx" sheet "tmp" and file "Axial-SRA_metadata_acc.xlsx" sheet "SRA_data"&nbsp;and extracted to csv<br />
- added conventional header with dataset name, PI name, version date<br />
- renamed columns to conform with BCO-DMO naming conventions (removed spaces)<br />
- combined environmental and SRA tables into one table; joined on sample_name and library_ID<br />
- removed unpopulated columns (ref_biomaterial, rel_to_oxygen, samp_collect_device, samp_mat_process, samp_size, host)<br />
- split lat_lon into lat and lon columns; changed sign of lon to negative&nbsp;to signify degrees west; reduced precision from variable to 4 decimal places<br />
- replaced commas with semi-colons</p>
Specified by the Principal Investigator(s)
asNeeded
7.x-1.1
Biological and Chemical Oceanography Data Management Office (BCO-DMO)
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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.
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MiSeq 300 x 300 bp PE
MiSeq 300 x 300 bp PE
PI Supplied Instrument Name: MiSeq 300 x 300 bp PE 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.
Beckman Coulter #A63881, Brea, CA
Beckman Coulter #A63881, Brea, CA
PI Supplied Instrument Name: Beckman Coulter #A63881, Brea, CA PI Supplied Instrument Description:Used to clean PCR products. Instrument Name: Coulter Counter Instrument Short Name: Instrument Description: An apparatus for counting and sizing particles suspended in electrolytes. It is used for cells, bacteria, prokaryotic cells and virus particles. A typical Coulter counter has one or more microchannels that separate two chambers containing electrolyte solutions.
from https://en.wikipedia.org/wiki/Coulter_counter
Niskin bottle
Niskin bottle
PI Supplied Instrument Name: Niskin bottle PI Supplied Instrument Description:Used to collect background seawater sample. Instrument Name: Niskin bottle Instrument Short Name:Niskin bottle Instrument Description: A Niskin bottle (a next generation water sampler based on the Nansen bottle) is a cylindrical, non-metallic water collection device with stoppers at both ends. The bottles can be attached individually on a hydrowire or deployed in 12, 24, or 36 bottle Rosette systems mounted on a frame and combined with a CTD. Niskin bottles are used to collect discrete water samples for a range of measurements including pigments, nutrients, plankton, etc. Community Standard Description: http://vocab.nerc.ac.uk/collection/L22/current/TOOL0412/
PI Supplied Instrument Name: Instrument Name: Thermal Cycler Instrument Short Name:Thermal Cycler Instrument Description: A thermal cycler or "thermocycler" is a general term for a type of laboratory apparatus, commonly used for performing polymerase chain reaction (PCR), that is capable of repeatedly altering and maintaining specific temperatures for defined periods of time. The device has a thermal block with holes where tubes with the PCR reaction mixtures can be inserted. The cycler then raises and lowers the temperature of the block in discrete, pre-programmed steps. They can also be used to facilitate other temperature-sensitive reactions, including restriction enzyme digestion or rapid diagnostics.
(adapted from http://serc.carleton.edu/microbelife/research_methods/genomics/pcr.html)
Cruise: NA108
NA108
E/V Nautilus
E/V Nautilus
vessel
NA108
Darlene Lim
National Aeronautics and Space Administration
E/V Nautilus
E/V Nautilus
vessel