|Created||October 5, 2016|
|Modified||January 17, 2017|
|State||Final no updates expected|
|Brief Description||Single amplified genomes (SAGs) of microbial cells isolated from Crab Spa, East Pacific Rise|
AT15-38: Samples for SAGs were obtained using the so-called 'titanium major samplers' (von Damm et al, 1985). Replicate samples of 1 ml aliquots of water were cryopreserved with 6% glycine betaine (Sigma) or 15% glycerol and stored at -80 ºC for the ”Single Cell” aliquot.
AT26-10: Background samples were obtained from the IGTs, in which the incubations were carried out. Just in this case, an aliquot was removed after sample retrieval and before starting the incubation. During AT26-10, samples were preserved with Gly-TE and stored at -80.
Cells were sorted, identified and sequenced by the Bigelow Laboratory Single Cell Genomics FacilityCenter (SCGC), following SCGCthe facilities’s standard practices: SCGC_Services_Description.pdf
On average, at least 5 million 2x150 bp or longer paired-end reads were generated per SAG using in-house MiSeq and NextSeq (Illumina) instruments. The obtained reads were pre-processed and, de novo, assembled and quality-controlled using algorithms SCGC's standard protocols that are optimized for single cell MDA products . A combination of tetramer homogeneity tests and blast searches against reference databases is used to detect potential DNA contaminants among the assembled contigs. Benchmark data demonstrating SCGC SAG WGS whole genome sequencing pipeline performance is available from the SCGC website http://data.bigelow.org/~scgc/WGS_benchmark_data/.
Genome annotation was performed through IMG (http://img-stage.jgi-psf.org/cgi-bin/submit/main.cgi).
MiSeq and NextSeq (Illumina) sequencers
General term for a laboratory instrument used for deciphering the order of bases in a strand of DNA. Sanger sequencers detect fluorescence from different dyes that are used to identify the A, C, G, and T extension reactions. Contemporary or Pyrosequencer methods are based on detecting the activity of DNA polymerase (a DNA synthesizing enzyme) with another chemoluminescent enzyme. Essentially, the method allows sequencing of a single strand of DNA by synthesizing the complementary strand along it, one base pair at a time, and detecting which base was actually added at each step.
Isobaric Gas Tight (IGT) samplers, designed and built by scientists and engineers at WHOI, are titanium instruments designed to be used with deep submergence vehicles to sample corrosive hydrothermal vent fluids at high temperature and high pressure. The IGT prevents the sampled fluid from degassing as pressure decreases during the vehicle’s ascent to the surface.
unique sample identification or number; any combination of alpha numeric characters; precise definition is file dependent
The origin of the sample used for the single cell sorting:
Background: Cell sorted from the natural water samples
Control: Cell sorted from the natural water samples incubated in isobaric chambers without any amendments
NO3-/H2, ~24C: Cell sorted from the natural water samples incubated at 24oC in isobaric chambers with the addition of NO3- (nitrate) and hydrogen (H2)
H2 only: Cell sorted from the natural water samples incubated at 24oC in isobaric chambers with the addition of hydrogen (H2)
NO3- only: Cell sorted from the natural water samples incubated at 24oC in isobaric chambers with the addition of NO3- (nitrate)hydrogen (H2)
O2, ~110uM: Cell sorted from the natural water samples incubated at 24oC in isobaric chambers with the addition of 110uM of O2 (oxygen)
NO3-/H2, ~50C: Cell sorted from the natural water samples incubated at 50oC in isobaric chambers with the addition of NO3- (nitrate) and hydrogen (H2)
Experimental conditions applied to experimental units. In comparative experiments, members of the complementary group, the control group, receive either no treatment or a standard treatment.
latitude, in decimal degrees, North is positive, negative denotes South; Reported in some datasets as degrees, minutes
longitude, in decimal degrees, East is positive, negative denotes West; Reported in some datsets as degrees, minutes
Link to an external data entry.
|Ramunas Stepanauskas||Bigelow Laboratory for Ocean Sciences (Bigelow)||✓|
|Nancy Copley||Woods Hole Oceanographic Institution (WHOI BCO-DMO)|
BCO-DMO Project Info
|Project Title||An Integrated Study of Energy Metabolism, Carbon Fixation, and Colonization Mechanisms in Chemosynthetic Microbial Communities at Deep-Sea Vents|
|Acronym||Microbial Communities at Deep-Sea Vents|
|Created||June 11, 2012|
|Modified||June 11, 2012|
Deep-sea hydrothermal vents, first discovered in 1977, are poster child ecosystems where microbial chemosynthesis rather than photosynthesis is the primary source of organic carbon. Significant gaps remain in our understanding of the underlying microbiology and biogeochemistry of these fascinating ecosystems. Missing are the identification of specific microorganisms mediating critical reactions in various geothermal systems, metabolic pathways used by the microbes, rates of the catalyzed reactions, amounts of organic carbon being produced, and the larger role of these ecosystems in global biogeochemical cycles. To fill these gaps, the investigators will conduct a 3-year interdisciplinary, international hypothesis-driven research program to understand microbial processes and their quantitative importance at deep-sea vents. Specifically, the investigators will address the following objectives: 1. Determine key relationships between the taxonomic, genetic and functional diversity, as well as the mechanisms of energy and carbon transfer, in deep-sea hydrothermal vent microbial communities. 2. Identify the predominant metabolic pathways and thus the main energy sources driving chemoautotrophic production in high and low temperature diffuse flow vents. 3. Determine energy conservation efficiency and rates of aerobic and anaerobic chemosynthetic primary productivity in high and low temperature diffuse flow vents. 4. Determine gene expression patterns in diffuse-flow vent microbial communities during attachment to substrates and the development of biofilms.
Integration: To address these objectives and to characterize the complexity of microbially-catalyzed processes at deep-sea vents at a qualitatively new level, we will pursue an integrated approach that couples an assessment of taxonomic diversity using cultivation-dependent and -independent approaches with methodologies that address genetic diversity, including a) metagenomics (genetic potential and diversity of community), b) single cell genomics (genetic potential and diversity of uncultured single cells), c) meta-transcriptomics and -proteomics (identification and function of active community members, realized potential of the community). To assess function and response to the environment, these approaches will be combined with 1) measurement of in situ rates of chemoautotrophic production, 2) geochemical characterization of microbial habitats, and 3) shipboard incubations under simulated in situ conditions (hypothesis testing under controlled physicochemical conditions). Network approaches and mathematical simulation will be used to reconstruct the metabolic network of the natural communities. A 3-day long project meeting towards the end of the second year will take place in Woods Hole. This Data Integration and Synthesis meeting will allow for progress reports and presentations from each PI, postdoc, and/or student, with the aim of synthesizing data generated to facilitate the preparation of manuscripts.
Intellectual Merit. Combining the community expression profile with diversity and metagenomic analyses as well as process and habitat characterization will be unique to hydrothermal vent microbiology. The approach will provide new insights into the functioning of deep-sea vent microbial communities and the constraints regulating the interactions between the microbes and their abiotic and biotic environment, ultimately enabling us to put these systems into a quantitative framework and thus a larger global context.
Broader Impacts. This is an interdisciplinary and collaborative effort between 4 US and 4 foreign institutions, creating unique opportunities for networking and fostering international collaborations. This will also benefit the involved students (2 graduate, several undergraduate) and 2 postdoctoral associates. This project will directly contribute to many educational and public outreach activities of the involved PIs, including the WHOI Dive & Discover program; single cell genomics workshops and Cafe Scientifique (Bigelow); REU (WHOI, Bigelow, CIW); COSEE and RIOS (Rutgers), and others. The proposed research fits with the focus of a number of multidisciplinary and international initiatives, in which PIs are active members (SCOR working group on Hydrothermal energy and the ocean carbon cycle, http://www.scorint. org/Working_Groups/wg135.htm; Deep Carbon Observatory at CIW, https://dco.gl.ciw.edu/; Global Biogeochemical Flux (GBF) component of the Ocean Observatories Initiative (OOI), http://www.whoi.edu/GBF-OOI/page.do?pid=41475)
|Dr Stefan M Sievert||Woods Hole Oceanographic Institution (WHOI)||Lead Principle Investigator||✓|