Table of Contents

• Coverage
• Dataset Description
  • Methods & Sampling
  • Data Processing Description
• Data Files
• Related Publications
• Parameters
• Instruments
• Deployments
• Project Information
• Program Information
• Funding

See SRA BioProject accession: PRJNA641911 (https://www.ncbi.nlm.nih.gov/bioproject/PRJNA641911) and BioSample accession IDs: SAMN15376334-SAMN15376347. Sequences are available via SRA (NCBI).

Sample collection from Axial Seamount:

From Fortunato et al (2018)

Sample collection from Axial Seamount took place in September 2013, August 2014, and August 2015. ROVs ROPOS and JASON were used to collect diffuse hydrothermal venting fluid. At each site, 3-L of diffuse venting fluid was pumped (100-150-ml per minute) onto a 0.22-um 47mm GWSP filter (Millipore). This fluid was collected using the Hydrothermal Fluid and Particle Sampler (HFPS; Butterfield et al. 2004), which was mounted on an ROV. The fluid intake for the HFPS has a temperature sensor to ensure a constant temperature during fluid collection. Filters were preserved in situ with RNALater. Background seawater was collected from a depth of 1500-m using a CTD mounted with 10-L Niskin bottles.

Extraction and sequencing for 18S tag-sequencing:

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.

BCO-DMO Processing Notes:
- data submitted in Excel files "Axial-Metagenome.environmental.1.0_JH.xlsx" sheet "tmp" and file "Axial-SRA_metadata_acc.xlsx" sheet "SRA_data" and extracted to csv
- added conventional header with dataset name, PI name, version date
- renamed columns to conform with BCO-DMO naming conventions (removed spaces)
- combined environmental and SRA tables into one table; joined on sample_name and library_ID
- removed unpopulated columns (ref_biomaterial, rel_to_oxygen, samp_collect_device, samp_mat_process, samp_size, host)
- split lat_lon into lat and lon columns; changed sign of lon to negative to signify degrees west; reduced precision from variable to 4 decimal places
- replaced commas with semi-colons

Adjusted C-DEBI Award Description:

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.

C-DEBI project link: https://www.darkenergybiosphere.org/award/probing-subseafloor-microbial-interactions-via-hydrothermal-vent-fluids-a-focus-on-protists/

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.