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

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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

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此資料的發布者及權利單位為 SCAR - Microbial Antarctic Resource System。 This work is licensed under a Creative Commons Attribution (CC-BY 4.0) License.

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此資源已向GBIF註冊，並指定以下之GBIF UUID: fe07fa3b-fe23-4a1d-aada-9d2c8fed883a。  SCAR - Microbial Antarctic Resource System 發佈此資源，並經由Scientific Committee on Antarctic Research同意向GBIF註冊成為資料發佈者。

關鍵字

Metadata

聯絡資訊

地理涵蓋範圍

Southern Ocean, Arctic Ocean and Indian ocean

分類群涵蓋範圍

Bacterial and eukaryote pelagic marine microorganisms <0.45μm

時間涵蓋範圍

計畫資料

無相關描述

參與計畫的人員:

取樣方法

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.

方法步驟描述:

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.

引用文獻

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).

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