Community diversity in microbial eukaryotes from lakes in the the Vestfold Hills, Antarctica

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Logares R, Tesson S, Canbäck B, Pontarp M, Hedlund K, Rengefors K (2019): Community diversity in microbial eukaryotes from lakes in the the Vestfold Hills, Antarctica. v1.1. SCAR - Microbial Antarctic Resource System. Dataset/Metadata. https://ipt.biodiversity.aq/resource?r=microbial_eukaryotes_lakes_from_vestfold_hills_antarctica&v=1.1

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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: 5f4ccd3e-5b1a-4145-aa43-94ecd45ced8a。  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

Vestfold Hills, East Antarctica The sampled lakes comprise: Lake Abraxas, Ace Lake, Crooked Lake, Deep Lake, Ekho Lake, Lake Hand, Highway Lake, Lebed Lake, Lake McNeil, Organic Lake, Pendant Lake, Rookery Lake, Lake Shield, Vereteno Lake, Lake Watts, and Lake Williams.

分類群涵蓋範圍

Eukaryotes (18S ssu rRNA, v4 region)

時間涵蓋範圍

計畫資料

The study aims at investigating the protist diversity in 17 aquatic locations in the region of the Vestfold hills, Eastern Antarctica (68°S, 78°E), including one marine site and 16 lakes, across a gradient of salinity spanning from 0 to 250 psu, and over the austral summer period (December 2008-February 2009).

參與計畫的人員:

取樣方法

Water samples from different depths were collected in 16 selected lakes as well as in one coastal marine site. Microbes were collected onto polycarbonate filters (Supor-200, 47mm; PALL Corporation, East Hills, NY, USA) and subsequently filters were stored at - 80°C.

方法步驟描述:

1. Community DNA from each sample was extracted from filter sections using a modified CTAB (Cetyl trimethyl ammonium bromide) extraction protocol in 2mL microfuge tubes along with 0.15g of beads from the PowerSoil™ DNA isolation kit (MOBio, California, USA). Each sample was incubated for 45min at 65°C in a mix of 700μL of CTAB, 24 μL β-mercaptoethanol, and 0.4 mg/mL of RNaseA (Sigma-Aldrich Sweden AB, Stockholm, Sweden), mixing by vortex agitation every 15 min. Samples were then ice-shocked and purified in 700μL of Chloroform:Isoamyl (24:1). After centrifugation (14,000g, 10 min, 4°C), the upper phase was removed and stored at -20°C for 60 min in one volume of isopropanol and a 0.5 volume of NaCl 5M. DNA was then precipitated through two consecutive centrifugations, first at14,000 g and 4°C during 30 min and afterwards, using the same setup during 15 min with 400 μL of ethanol 75%. After removing the supernatant, DNA pellets were dried using a speed vacuum DNA dryer at 300 g, 43°C, during 20 min. Pellets were then dissolved in 40-100 μL of milliQ water. The quality and quantity of DNA was measured using agarose gels (1.5%, TBE 0.5x) as well as a Nanodrop 2000/2000C Spectrophotometer v1.0 (Thermo Scientific, NanoDrop products, Delaware, USA).
2. Polymerase chain reactions (PCR) were performed in 96 wells plates. For each template DNA, four to nine replicate PCRs were performed following Logares et al. (2012), and subsequently combined. The PCR mix comprised 12.5 μL of 2x Phusion® GC Master Mix (F-532L, Finnzymes Oy, Vantaa, Finland), 4% of DMSO, 10 pmol μL-1 of barcoded fusion primers (Invitrogen™, Life Technologies Europe BV, Stockholm, Sweden), and 5 ng of DNA template in a final volume of 25μL that was adjusted with distilled water. Universal primers adapted from Stoeck et al. (2010) were used to amplify fragments of about 380bp from the V4 18S rDNA region and subsequently sequenced using 454 pyrosequencing. Primers included adapters for uni-directional (library Lib-L) sequencing. The forward primer comprised, in the 5’-3’ direction, a 454-specific adapter, followed by a multiplex identifier (MID, 7bp) and the template-specific primer. The reverse primer included a 454-specific adapter and the template-specific primer. PCR amplification, including number of cycles, was performed as described in Logares et al (2012). Quality control and quantification of PCR products were performed using agarose gels (1.5%, TBE 0.5x). PCR products were purified using the QIAquick® PCR purification Kit (QIAGEN GmbH, Hilden, Germany) and eluted in 45 µl of water (molecular biology grade). Amplicon concentration was estimated using Nanodrop (Thermo Scientific, Wilmington, USA).
3. 454-pyrosequencing was conducted at the Lund University Sequencing Facility. Two pools of fourteen samples, containing 333.3 ng of DNA each, were produced. In each pool, short fragments were removed using magnetic beads (Agencourt AMPure XP; Beckman Coulter AB, Bromma, Sweden). Pools were subsequently inspected using a DNA 1000 kit on a 2100 Bioanalyzer (Agilent Technologies Sweden AB, Kista, Sweden). Amplicons were quantified using the Quant-iT dsDNA assay kit (Invitrogen) and a Quantiflour fluorometer (Promega Biotech AB, Nacka, Sweden). Pools were diluted to obtain a total of 1×107 copies µL-1. Titration and library production (aiming at 10-15% recovery) were performed using emulsion PCR with the Lib-L kit (Roche AB, Stockholm, Sweden) for unidirectional sequencing. DNA positive beads were enriched, counted on an Innovatis CASY particle counter (Roche), processed using the XLR70 sequencing kit (Roche), and loaded onto a picotiter plate for pyrosequencing on a GS FLX Titanium platform (Roche).

引用文獻

1. Logares, R., Tesson, S. V., Canbäck, B., Pontarp, M., Hedlund, K., & Rengefors, K. (2018). Contrasting prevalence of selection and drift in the community structuring of bacteria and microbial eukaryotes. Environmental microbiology.

額外的詮釋資料