virome_antarctic_lakes_2010

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Les chercheurs doivent citer cette ressource comme suit:

de Carcer D A, Lopez-Bueno A, Alonso-Lobo J, Quesada A, Alcami A (2021): virome_antarctic_lakes_2010. v1.0. SCAR - Microbial Antarctic Resource System. Dataset/Metadata. https://ipt.biodiversity.aq/resource?r=virome_antarctic_lakes_2010&v=1.0

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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.

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Cette ressource a été enregistrée sur le portail GBIF, et possède l'UUID GBIF suivante : 9c226382-bbf8-420c-bfcc-bb417be1f181.  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

Antarctic Peninsula: Caleta Cierva, Avian Island, Green Island, Pourquoi Pas Island and Livingston Island (South Shetland Islands)

Couverture temporelle

Données sur le projet

Pas de description disponible

Les personnes impliquées dans le projet:

Méthodes d'échantillonnage

Water samples were preserved at 4°C and viral particles purified within the first 24 h of sampling. From each water body, 60–90 l were first filtered through a 30 μm nylon mesh to remove large particles, and then by tangential flow filtration (TFF) using a peristaltic pump coupled to a Centramate holder (Pall) with two cassettes of 0.45 μm (2 × 0.019 m2, PES) to remove bacteria and smaller eukaryotes.

Description des étapes de la méthode:

1. The filtrated viral fraction was concentrated 100 times by TFF with two cassettes of 70 kDa exclusion size at pressures below 10 p.s.i., in order to maintain viral particle integrity. Viral stocks were preserved at −20°C for transportation, and finally stored at −80°C before subsequent processing. All material was acid-rinsed (0.1 N HCl) and extensively washed with Milli Q or 70 kDa-filtered lake water.
2. Frozen stocks were thawed at 4°C with gentle rocking, and then passed through a 25% sucrose cushion by centrifugation for 16 h at 60 000 g and 4°C. The resulting pellets were resuspended in TE buffer (10 mM Tris pH 8, 1 mM EDTA), and filtered using a sterile 0.45 μm syringe filter. Viral concentrates were then treated with DNAse I (500 U ml−1), Nuclease S7 (500 U ml−1), RNAse A (100 μg ml−1) and RNAse H (2 U per reaction) for 30 min at room temperature to remove free nucleic acids. Nuclease reactions were stopped with EDTA and EGTA (12 and 2 mM, respectively), and viral capsids and envelopes were then disrupted with SDS (0.5%) and proteinase K (200 μg ml−1) treatment.
3. Viral DNA was extracted with phenol–chloroform, ethanol precipitated and randomly amplified using Phi29 polymerase and modified random hexamers (GenomiPhi HY, GE Healthcare) for 2.5 h, according to the manufacturer's instructions. The samples were subsequently shot-gun sequenced with a Roche 454 FLX sequencer (Parque Científico de Madrid).

Citations bibliographiques

1. de Cárcer A. D., López-Bueno A., Alonso-Lobo J. M., Quesada A., & Alcamí A. (2016). Metagenomic analysis of lacustrine viral diversity along a latitudinal transect of the Antarctic Peninsula. FEMS microbiology ecology, 92(6), fiw074.

Métadonnées additionnelles