RNA-Virome (RNA shotgun sequencing) from lake Limnopolar on Livingston Island (Antarctica)

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¿Cómo referenciar?

Los usuarios deben citar este trabajo de la siguiente manera:

Lopez-Bueno A, Rastrojo A, Peiro R, Arenas M, Alcami A (2019): RNA-Virome (RNA shotgun sequencing) from lake Limnopolar on Livingston Island (Antarctica). v1.0. SCAR - Microbial Antarctic Resource System. Dataset/Metadata. https://ipt.biodiversity.aq/resource?r=virome_livingston_island_antarctica&v=1.0

Derechos

Los usuarios deben respetar los siguientes derechos de uso:

El publicador y propietario de los derechos de este trabajo es SCAR - Microbial Antarctic Resource System. Esta obra está bajo una licencia Creative Commons de Atribución/Reconocimiento (CC-BY 4.0).

Registro GBIF

Este recurso ha sido registrado en GBIF con el siguiente UUID: 9b846f85-327e-412d-abb7-2eef49b6fae4.  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

Cobertura geográfica

Limnopolar lake, Livingston Island, South Shetland Islands, Antarctica

Cobertura taxonómica

RNA-Viruses targeted with RNA-shotgun sequencing (454 pyrosequencing)

Cobertura temporal

Datos del proyecto

No hay descripción disponible

Personas asociadas al proyecto:

Métodos de muestreo

Samples were taken aseptically.

Descripción de la metodología paso a paso:

1. Cyanobacterial mat samples (2.5 g) were homogenized in SM buffer (50 mm Tris‐HCl pH 7.5, 100 mm NaCl and 8 mm MgSO4.7H2O) by three cycles of vigorous vortex and sonication in a water bath during 20 and 10 s, respectively, then centrifuged at 3000 g for 5 min. This process was repeated twice. The resulting supernatants were combined and centrifuged at 8000 g for 1 h and then filtered through a 0.45‐μm syringe filter (Millex, Durapore PVDF) to remove cellular organisms. The resulting viral fractions, as well as those from lake water samples, were purified as described previously (López‐Bueno et al. 2009). In the case of the water sample collected in 2010, 0.45 μm filtration was carried out by TFF using two 0.093 m2 polyethersulfone filter cassettes (Pall) and nuclease treatment of purified viral particles included 100 U/mL of nuclease S7 (Roche). RNA viral genomes were purified with Trizol‐LS (Invitrogen) followed by DNAseI RNAse‐free (Roche) treatment before randomly amplification by sequence independent single primer amplification (SISPA) (Victoria et al. 2008; Djikeng et al. 2009; Culley et al. 2010). Briefly, Superscript II or III and the Klenow fragment (3′–>5′exo‐) enzyme (NEBiolabs) were used to convert RNA into dsDNA using 60 pmol of pseudo‐degenerated primers FR26‐RV (5′‐GCCGGAGCTCTGCAGATATCNNNNNN‐3) for the water sample collected in 2007 and primer A (5′‐GTTTCCCAGTCACGATANNNNNNNNN‐3) for samples collected in 2006 and 2010. After 40 cycles of PCR amplification with FastStart high fidelity polymerase (Roche), DNA fragments between 500 and 3000 bp were gel‐extracted with QIAquick Gel Extraction kit (Qiagen) and sequenced in the 454 GS FLX titanium platforms (Roche‐454) from LifeSequencing (Valencia, Spain) or from Parque Científico de Madrid (Spain).

Referencias bibliográficas

1. López‐Bueno, A., Rastrojo, A., Peiró, R., Arenas, M., & Alcamí, A. (2015). Ecological connectivity shapes quasispecies structure of RNA viruses in an Antarctic lake. Molecular ecology, 24(19), 4812-4825. https://doi.org/10.1111/mec.13321

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