8d3fae22-72bd-4e8f-adcd-7f57079c87ce https://ipt.biodiversity.aq/resource?r=pole_to_pole_marine_bacterial_communities Surface and deep marine bacterial communities in the Arctic and Antarctic Jean-François Ghiglione University Pierre et Marie Curie
FR
Pierre Galand University Pierre et Marie Curie
FR
Thomas Pommier Institut National de la Recherche Agronomique (INRA)
Villeurbanne FR
Carlos Pedros-Alio Institut de Ciències del Mar
Barcelona ES
Elizabeth Maas National Institute of Water and Atmospheric Research
Wellington NZ
Kevin Bakker University of Georgia
Athens US
Stefan Bertilson Uppsala University
Uppsala SE
David Kirchman University of Delaware
Lewes US
Connie Lovejoy Université Laval
Québec CA
Patricia Yager University of Georgia
Athens US
Alison Murray Desert Research Institute
Reno US
Maxime Sweetlove Royal Belgian Institute for Natural Sciences Research assistent
Rue Vautier 29 Brussels 1000 BE
msweetlove@naturalsciences.be
user 2019-03-19 eng 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). Metadata GBIF Dataset Type Vocabulary: http://rs.gbif.org/vocabulary/gbif/dataset_type.xml This work is licensed under a Creative Commons Attribution (CC-BY) 4.0 License. The Arctic Ocean and the Southern Ocean -159.34 126 79.99 -73.96 Bacteria (16S ssu rRNA gene, v6 region) domain Bacteria Bacteria unkown Jean-François Ghiglione University Pierre et Marie Curie
FR
Alison Murray Desert Research Institute
Reno US
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). Water samples were taken from the Southern and the Arctic Oceans Samples were collected with a 5 l Niskin bottle. International Census of Marine Microbes Linda Amaral-Zettler The role of the International Census of Marine Microbes (ICoMM) is to promote an agenda and an environment that will accelerate discovery, understanding, and awareness of the global significance of marine microbes. More details can be found in: 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. Funding to support sample collection was provided by the Institut Français pour la Recherche et la Technologie Polaires; the Spanish Ministry of Education and Science; the New Zealand International Polar Year-Census of Antarctic Marine Life Project [Phases 1 (So001IPY) and 2 (IPY2007-01)); the Natural Sciences and Engineering Council (NSERC) of Canada; National Science Foundation Grants OPP-0124733, ANT-0632389, and ANT-0741409; and the Swedish Polar Research Secretariat. Pyrosequencing was provided by the International Census of Marine Microbes (ICoMM) with financial support from a W. M. Keck Foundation award to the Marine Biological Laboratory in Woods Hole.
2019-01-29T04:25:16.621+01:00 dataset Ghiglione J, Galand P, Pommier T, Pedros-Alio C, Maas E, Bakker K, Bertilson S, Kirchman D, Lovejoy C, Yager P, Murray A (2019): Surface and deep marine bacterial communities in the Arctic and Antarctic. v1.1. SCAR - Microbial Antarctic Resource System. Dataset/Metadata. https://ipt.biodiversity.aq/resource?r=pole_to_pole_marine_bacterial_communities&v=1.1 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. 8d3fae22-72bd-4e8f-adcd-7f57079c87ce/v1.1.xml