Antarctic Surface Snow Bacterial Communities

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Comment citer

Les chercheurs doivent citer cette ressource comme suit:

Malard L, Sabacka M, Magiopoulos I, Mowlem M, Hodson A, Tranter M, Siegert M, Pearce D (2019): Antarctic Surface Snow Bacterial Communities. v1.0. SCAR - Microbial Antarctic Resource System. Dataset/Metadata. https://ipt.biodiversity.aq/resource?r=antarctic_surface_snow_bacterial_communities&v=1.0

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 : f9644d4b-3c39-4147-9d84-4baff93e1737.  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 region between the South Orkney Islands and the Ellsworth Mountains (Antarctica)

Couverture taxonomique

Bacteria were profiled by targeting the v3-v4 region of the 16S ssu rRNA gene with the primers 341F and 785R

Données sur le projet

Pas de description disponible

Les personnes impliquées dans le projet:

Méthodes d'échantillonnage

Snow was collected from the surface to represent collection during melt water production for hot-water drilling. At each sampling location, a 1 m snow pit was excavated and the top 30 cm of snow was sampled using an ethanol sterilized shovel and Whirl-Pak bags (Nasco, WI, United States). Samples were transported to the field laboratory, where they were melted and passed through 0.2 μm Sterivex filters (Merck, Darmstadt, Germany), before freezing at −20°C for transport and processing in the United Kingdom.

Description des étapes de la méthode:

1. SkyBlu, Pine Island Bay and Ellsworth snow samples were processed in two ways; PMA-treated and non-PMA-treated in order to differentiate the potentially viable microbial community from relic DNA. Each 0.2 μm filter was cut in half using sterile and DNAase/ethanol treated razors. For each sample, one-half was processed with PMA as per Nocker and Camper (2009) and Fittipaldi et al. (2012), using a 20 mM stock solution of PMA (Biotium, Hayward, CA, United States) in a 20% (v/v) aqueous solution of dimethyl sulfoxide (DMSO). Filters for PMA treatment were placed in a 6-well plate and a PMA solution at a final concentration of 100 μM was added. Cross-linking was initiated by 10 min incubation on ice, in the dark with occasional mixing, followed by 5 min of light exposure using a 650 W halogen lamp (FLASH 2000 L, DTS, Italy), at a 20 cm distance. The process was carried out in a laminar flow hood to avoid contamination of the samples. Non-PMA-treated samples were incubated in a 20% (v/v) aqueous solution of DMSO, and treated following the same protocol as PMA-treated samples. All samples were washed twice with sterile phosphate buffered saline (PBS) and all samples were then used for DNA extraction. DNA from snow samples was extracted using the PowerWater kit from MoBio (Qiagen, Carlsbad, CA, United States). Each sample was PCR amplified using the primers 341F and 785R covering the V3-V4 regions of the 16S rRNA gene (Klindworth et al., 2013), under the following conditions: initial denaturation at 95°C for 5 min then 25 cycles of 40 s denaturation at 95°C; primer annealing at 55°C for 2 min; and elongation at 72°C for 1 min then a final elongation at 72°C for 7 min (Hodson et al., 2017). DNA extraction kit controls were included alongside the snow derived DNA and sequenced. PCR amplicons were cleaned, normalized, quantified and supplemented with 5% PhiX before being loaded on Illumina MiSeq, as per the Illumina standard operating protocol (Kozich et al., 2013).
2. DNA from snow samples was extracted using the PowerWater kit from MoBio (Qiagen, Carlsbad, CA, United States). Each sample was PCR amplified using the primers 341F and 785R covering the V3-V4 regions of the 16S rRNA gene, under the following conditions: initial denaturation at 95°C for 5 min then 25 cycles of 40 s denaturation at 95°C; primer annealing at 55°C for 2 min; and elongation at 72°C for 1 min then a final elongation at 72°C for 7 min (Hodson et al., 2017). DNA extraction kit controls were included alongside the snow derived DNA and sequenced. PCR amplicons were cleaned, normalized, quantified and supplemented with 5% PhiX before being loaded on Illumina MiSeq, as per the Illumina standard operating protocol (Kozich et al., 2013).

Citations bibliographiques

1. Malard, L. A., Šabacká, M., Magiopoulos, I., Hodson, A., Tranter, M., Siegert, M. J., & Pearce, D. A. (2019). Spatial variability of Antarctic surface snow bacterial communities. Frontiers in Microbiology, 10, 461. https://doi.org/10.3389/fmicb.2019.00461

Métadonnées additionnelles