Surface and deep marine bacterial communities in the Arctic and Antarctic
Citation
Ghiglione J, Galand P, Pommier T, Pedros-Alio C, Maas E, Bakker K, Bertilson S, Kirchman D, Lovejoy C, Yager P, Murray A, Sweetlove M (2019). Surface and deep marine bacterial communities in the Arctic and Antarctic. Version 1.1. SCAR - Microbial Antarctic Resource System. Metadata dataset https://doi.org/10.15468/yxczuk accessed via GBIF.org on 2024-09-01.Description
Amplicon sequencing dataset (454 pyrosequencing) of Bacteria (16S ssu rRNA gene, v6 region) in surface and deep waters of the Arctic and the Southern oceans. This dataset is part of the International Census of Marine Microbes (ICoMM).Sampling Description
Study Extent
Water samples were taken from the Southern and the Arctic OceansSampling
Samples were collected with a 5 l Niskin bottle.Method steps
- DNA extraction buffer (0.1 M Tris-HCl [pH 8], 0.1 M Na-EDTA [pH 8], 0.1 M NaH2PO4 [pH 8], 1.5 M NaCl, 5% cetyltri- methylammonium bromide), and proteinase K (1%) was added to each filter. Samples were frozen at -80°C and thawed at 65°C three times and then incubated on a rotating carousel for 30 min at 37°C. Sodium dodecyl sulfate (SDS; 20%) was added to each sample, and the samples were incubated at 65°C on a rotating carousel for 2 h. The liquid was then removed from the filters using a 3-ml syringe and placed in a 2-ml microcentrifuge tube, which was centrifuged at room temperature (6,000 g; 5 min). The supernatant from each microcentrifuge tube was then placed in separate 15-ml Falcon collection tubes. DNA extraction buffer, lysozyme (200 ul; 50 mg ml), SDS, and proteinase K were then added to each filter (1 ml and 75 and 20 ul, respectively) and to each microcentrifuge tube containing spun-down particles (0.37 ml and 75 and 10 ul, respectively). Both the filter samples and the microcentrifuge tubes were incubated on a rotating carousel for 10 min. The microcentrifuge tubes were again centrifuged (6,000 g; 5 min), and the supernatant was added to the appropriate collection tube. Liquid was then removed from the filters, placed in the microcentrifuge tubes, and centrifuged (6,000 g; 5 min), and the supernatant was added to the collection tubes. The extraction buffer, SDS, and proteinase K were added to each filter and the particles again, and the extraction process was repeated. An equal volume of phenol:chloroform:isoamyl alcohol step (25:24:1) was added to each collection tube of supernatant, and the tubes were vortexed and centri- fuged (1,200 g; 10 min). The aqueous (top) layer from each tube was drawn off into a 30-ml acid-washed sterile Corex (Corning) tube, and an equal volume of isopropanol was added to each tube and mixed gently. Often additional aliquots of isopropanol-water (1:1) were added to adequately dissolve the aqueous layer in the isopropanol. After the tubes were incubated for 1 h at room temperature, the precipitated DNA was centrifuged at room temperature (16,000 g; 20 min), and the isopropanol supernatant was removed and replaced with 5 ml of 70% ethanol. After a final centrifugation (16,000 g; 20 min), the ethanol was removed and the DNA was dried down and resuspended in 95 ul of TE buffer (10 mM Tris 1 mM EDTA, pH 8.0). The DNA was purified using Qiaquick PCR purification columns (Qiagen) according to the manufacturer’s instructions and stored at 20°C.
- PCR amplicon was done by adding genomic DNA (3–10 ng) to three separate 30 ul amplification mixes. The amplification mix contained 5 units of Pfu Turbo polymerase (Stratagene, La Jolla, CA), 1 Pfu reaction buffer, 200 uM dNTPs (Pierce Nucleic Acid Technologies, Milwaukee, WI), and a 0.2 uM concentration of each primer in a volume of 100 ul. Cycling conditions were an initial denaturation at 94°C for 3 min; 30 cycles of 94°C 30 s, 57°C for 45 s, and 72°C for 1 min; and a final 2-min extension at 72°C. The products were pooled after cycling and cleaned by using the MinElute PCR purification kit (Qiagen, Valencia, CA). The quality of the product was assessed on a Bioanalyzer 2100 (Agilent, Palo Alto, CA) using a DNA1000 LabChip. Only sharp, distinct amplification products with a total yield of 200 ng were used for 454 sequencing. The fragments in the amplicon libraries were bound to beads under conditions that favor one fragment per bead. The beads were emulsified in a PCR mixture in oil, and PCR amplification occurred in each droplet, generating 10 million copies of a unique DNA template. After breaking the emulsion, the DNA strands were denatured, and beads carrying single- stranded DNA clones were deposited into wells on a PicoTiter- Plate (454 Life Sciences) for pyrosequencing on a Genome Sequencer 20 system (Roche, Basel, Switzerland).
Taxonomic Coverages
Bacteria (16S ssu rRNA gene, v6 region)
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Bacteriacommon name: Bacteria rank: domain
Geographic Coverages
The Arctic Ocean and the Southern Ocean
Bibliographic Citations
- Amaral-Zettler, L., Artigas, L.F., Baross, J., Bharathi, L., Boetius, A., Chandramohan, D., Herndl, G., Kogure, K., Neal, P., Pedros-Alio, C., Ramette, A., Schouten, S., Stal, L., Thessen, A., de Leeuw, J. & Sogin, M. 2010. A global census of marine microbes, In: Life in the World's Oceans: Diversity, Distribution and Abundance, Blackwell Publishing Ltd., Oxford, (Ed. McIntyre), pp. 223-45. -
- Ghiglione, J. F., Galand, P. E., Pommier, T., Pedrós-Alió, C., Maas, E. W., Bakker, K., ... & Murray, A. E. (2012). Pole-to-pole biogeography of surface and deep marine bacterial communities. Proceedings of the National Academy of Sciences, 201208160. -
Contacts
Jean-François Ghiglioneoriginator
University Pierre et Marie Curie
FR
Pierre Galand
originator
University Pierre et Marie Curie
FR
Thomas Pommier
originator
Institut National de la Recherche Agronomique (INRA)
Villeurbanne
FR
Carlos Pedros-Alio
originator
Institut de Ciències del Mar
Barcelona
ES
Elizabeth Maas
originator
National Institute of Water and Atmospheric Research
Wellington
NZ
Kevin Bakker
originator
University of Georgia
Athens
US
Stefan Bertilson
originator
Uppsala University
Uppsala
SE
David Kirchman
originator
University of Delaware
Lewes
US
Connie Lovejoy
originator
Université Laval
Québec
CA
Patricia Yager
originator
University of Georgia
Athens
US
Alison Murray
originator
Desert Research Institute
Reno
US
Maxime Sweetlove
metadata author
position: Research assistent
Royal Belgian Institute for Natural Sciences
Rue Vautier 29
Brussels
1000
BE
email: msweetlove@naturalsciences.be
Jean-François Ghiglione
administrative point of contact
University Pierre et Marie Curie
FR
Alison Murray
administrative point of contact
Desert Research Institute
Reno
US