Pelagic microbes (Eukaryote 18S and Material 16S amplicons) from sites in the Southern, Indian and Arctic Oceans

Versions

The table below shows only published versions of the resource that are publicly accessible.

How to cite

Researchers should cite this work as follows:

Schroeder D, Lebret K, Balestreri C, Highfield A, Schroeder J, Thorpe S, Moore K, Pasckiewicz K, Pfaff M, Rybicki E, Flaviani F (2018): Pelagic microbes (Eukaryote 18S and Material 16S amplicons) from sites in the Southern, Indian and Arctic Oceans. v1.2. SCAR - Microbial Antarctic Resource System. Dataset/Metadata. https://ipt.biodiversity.aq/resource?r=pelagic_microbes_chlorophyll_maximum_southern_indian_arctic_oceans&v=1.2

Rights

Researchers should respect the following rights statement:

The publisher and rights holder of this work is SCAR - Microbial Antarctic Resource System. This work is licensed under a Creative Commons Attribution (CC-BY 4.0) License.

GBIF Registration

This resource has been registered with GBIF, and assigned the following GBIF UUID: fe07fa3b-fe23-4a1d-aada-9d2c8fed883a.  SCAR - Microbial Antarctic Resource System publishes this resource, and is itself registered in GBIF as a data publisher endorsed by Scientific Committee on Antarctic Research.

Keywords

Metadata

Contacts

Geographic Coverage

Southern Ocean, Arctic Ocean and Indian ocean

Taxonomic Coverage

Bacterial and eukaryote pelagic marine microorganisms <0.45μm

Temporal Coverage

Project Data

No Description available

The personnel involved in the project:

Sampling Methods

At each station, 1 l of seawater from the chlorophyll maximum layer was sampled by a conductivity temperature depth (CTD) rosette sampler on-board the R/V Roger Revelle. An aliquot of 250 ml was filtered through a 0.45-μm polycarbonate filter. DNA extraction of material retained on the filter was performed using the Qiagen DNeasy Blood and Tissue protocol (QIAGEN, Valencia, CA, United States). The DNA was stored at −21°C and subsequently transferred to Plymouth, United Kingdom, for further processing. An additional 50 ml sample of filtered water was stored at 4°C in the dark for further processing in the laboratory after the cruise.

Method step description:

1. For Bacteria, the V4 region of 16S ribosomal RNA gene was amplified using the universal primer pair 515F/806R and Illumina tagged primers. Eukaryotes were characterized using the 18S ribosomal RNA gene, using primer pair 1391F/EukB, and Illumina tagging to amplify the V9 region.
2. First, a real-time PCR was run for each sample to determine the mid-exponential threshold of each reaction. For all PCRs, 1–5 μl of DNA, corresponding to 1.47–38.52 ng/μl, respectively, were added to 5× Colorless GoTaq Flexi Buffer (Promega, Madison, WI, United States), 1.5 μl MgCl2 Solution 25 mM (Promega, Madison, WI, United States), 2.5 μl dNTPs (10 mM final concentration, Promega, Madison, WI, United States), 1 μl Evagreen Dye 20× (Biotium, Fremont, CA, United States), 0.1 μl GoTaq DNA Polymerase (5 U/μl – Promega, Madison, WI, United States), and sterile water was added to reach the final volume of 25 μl for each reaction. The PCRs were run on a Corbette Rotor-Gene 6000 (QIAGEN, Valencia, CA, United States), with initial denaturation at 94°C for 3 min, followed by 40 cycles of a three step PCR: 94°C for 45 s, 50°C for 60 s, and 72°C for 90 s. Fluorescence in the green channel was recorded at the end of each annealing/extension step. The cycle threshold of the amplification in the exponential phase was recorded for each sample.
3. Each real time PCR was carried out in triplicate on a unique aliquot of DNA subsampled from the same extraction, and sequenced using single end reads. Next, a standard PCR amplification was carried out in triplicate and run with the same conditions as the first real-time PCR, excluding the addition of the Evagreen Dye, until the previously determined cycle threshold was reached. PCR products were then run on a 1.4% agarose gel to confirm the success of the amplification and the product size of the amplification. The bands were cut from the gel and purified using the Zymoclean Gel DNA Recovery Kit (Zymo Research, Irvine, CA, United States). Quantity and quality were verified with a NanoDrop 1000 (Thermo Fisher Scientific, Wilmington, DE, United States) and QuantiFluor E6090 (Promega, Madison, WI, United States). The PCR products were combined in equimolar concentrations as measured on the Bioanalyzer (Agilent Technologies, Cheshire, United Kingdom). The final pooled samples were denatured and diluted to 6 pM and mixed with 1 pM PhiX control (Illumina, San Diego, CA, United States), read 1 sequencing primer was diluted in HT1, before the flowcell was clustered on the cBOT (Illumina, San Diego, CA, United States). Multiplexing sequencing primers and read 2 sequencing primers were mixed with Illumina HP8 and HP7 sequencing primers, respectively. The flowcell was sequenced (100 pair end-PE) on HiSeq 2500 using SBS reagents v3.

Bibliographic Citations

1. Flaviani, F., Schroeder, D., Lebret, K., Balestreri, C., Schroeder, J., Moore, K., ... & Rybicki, E. (2018). Distinct oceanic microbiomes (from viruses to protists) found either side of the Antarctic Polar Front. Frontiers in Microbiology, 9, 1474.
2. Flaviani, F. (2017). Microbial biodiversity in the southern Indian Ocean and Southern Ocean (Doctoral dissertation, University of Cape Town).

Additional Metadata