Surface and deep marine bacterial communities in the Arctic and Antarctic

Versions

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Comment citer

Les chercheurs doivent citer cette ressource comme suit:

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

Droits

Les chercheurs doivent respecter la déclaration de droits suivante:

L’éditeur et détenteur des droits de cette ressource est SCAR - Microbial Antarctic Resource System. Ce travail est sous licence Creative Commons Attribution (CC-BY) 4.0.

Enregistrement GBIF

Cette ressource a été enregistrée sur le portail GBIF, et possède l'UUID GBIF suivante : 8d3fae22-72bd-4e8f-adcd-7f57079c87ce.  SCAR - Microbial Antarctic Resource System publie cette ressource, et est enregistré dans le GBIF comme éditeur de données avec l'approbation du Scientific Committee on Antarctic Research.

Mots-clé

Metadata

Contacts

Couverture géographique

The Arctic Ocean and the Southern Ocean

Couverture taxonomique

Bacteria (16S ssu rRNA gene, v6 region)

Données sur le projet

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.

Les personnes impliquées dans le projet:

Méthodes d'échantillonnage

Samples were collected with a 5 l Niskin bottle.

Description des étapes de la méthode:

1. 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.
2. 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).

Citations bibliographiques

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.
2. 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.

Métadonnées additionnelles