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

Última versión Publicado por SCAR - Microbial Antarctic Resource System en Mar 19, 2019 SCAR - Microbial Antarctic Resource System

Amplicon sequencing dataset targeting Eukaryotes (18S V9 region) and Bacteria (16S V4 region) in the <0.45μm fraction of pelagic microbes, sampled in the chlorophyll maximum zone.

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¿Cómo referenciar?

Los usuarios deben citar este trabajo de la siguiente manera:

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

Derechos

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El publicador y propietario de los derechos de este trabajo es SCAR - Microbial Antarctic Resource System. This work is licensed under a Creative Commons Attribution (CC-BY) 4.0 License.

Registro GBIF

Este recurso ha sido registrado en GBIF con el siguiente UUID: fe07fa3b-fe23-4a1d-aada-9d2c8fed883a.  SCAR - Microbial Antarctic Resource System publica este recurso, y está registrado en GBIF como un publicador de datos avalado por Scientific Committee on Antarctic Research.

Palabras Clave

Metadata

Contactos

¿Quién creó el recurso?:

Declan Schroeder
Marine Biological Association of the United Kingdom Plymouth GB
Karen Lebret
Marine Biological Association of the United Kingdom Plymouth GB
Cecilia Balestreri
Marine Biological Association of the United Kingdom Plymouth GB
Andrea Highfield
Marine Biological Association of the United Kingdom Plymouth GB
Joanna Schroeder
Marine Biological Association of the United Kingdom Plymouth GB
Sally Thorpe
British Antarctic Survey Cambridge GB
Karen Moore
Exeter Sequencing Service Exeter GB
Konrad Pasckiewicz
Department of Environmental Affairs Cape Town ZA
Maya Pfaff
Department of Environmental Affairs Cape Town ZA
Edward Rybicki
University of Cape Town Cape Town ZA
Flavia Flaviani
University of Cape Town Cape Town ZA

¿Quién puede resolver dudas acerca del recurso?:

Flavia Flaviani
University of Cape Town Cape Town ZA

¿Quién documentó los metadatos?:

Maxime Sweetlove
Research assistent
Royal Belgian Institute of Natural Sciences Rue Vautier 29 1000 Brussels BE

¿Quién más está asociado con el recurso?:

Usuario

Cobertura Geográfica

Southern Ocean, Arctic Ocean and Indian ocean

Coordenadas límite Latitud Mínima Longitud Mínima [-58.71, 25.382], Latitud Máxima Longitud Máxima [74.09, 113.4]

Cobertura Taxonómica

Bacterial and eukaryote pelagic marine microorganisms <0.45μm

Dominio  Bacteria (Bacteria),  Eukaryota (Eukaryotes)

Cobertura Temporal

Fecha Inicial / Fecha Final 2012-03-06 / 2013-04-14

Datos del Proyecto

No hay descripción disponible

Título South African National Research Foundation (NRF) grant to ER (CPR20110717000020991)
Fuentes de Financiación The project was funded by a South African National Research Foundation (NRF) grant to ER (CPR20110717000020991), the FP7-OCEAN-2011 call, MicroB3 (grant number 287589) and the NERC eDNA award (grant number NE/N006151/1), and the Ocean Ecosystems Program at the British Antarctic Survey (NERC, United Kingdom).

Personas asociadas al proyecto:

Flavia Flaviani

Métodos de Muestreo

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.

Área de Estudio Point measurement samples were collected during the Great Southern Coccolithophore Belt expedition (GSCB-cruise RR1202). Stations S1 and S2 were located in the South-West Indian Ocean, stations S3 and S4 in the Southern Ocean, and stations S5 and S6 in the South-East Indian Ocean.

Descripción de la metodología paso a paso:

  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.

Referencias Bibliográficas

  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).

Metadatos Adicionales

Identificadores Alternativos fe07fa3b-fe23-4a1d-aada-9d2c8fed883a
https://ipt.biodiversity.aq/resource?r=pelagic_microbes_chlorophyll_maximum_southern_indian_arctic_oceans