Dataset: Montastraea annularis white plague viromes and 16S amplicons
Deployment: Vega_Thurber_Coral_Virus

Sampling of the scleractinian coral, M. annularis, was conducted at Brewers Bay, in St. Thomas, US Virgin Islands, during a concurrent WP outbreak and bleaching event in September 2010. Collections took place over 2 days at depths of 5.5–7.6 m. Temperature at depth was 29.0 degrees C. All M. annularis colonies used in this study were located within ~75 m of each other. Coral samples were collected using SCUBA, where two to three cores of tissue attached to skeleton were removed from each M. annularis colony using a 2 cm diameter corer and hammer (USVI Department of Planning and Natural Resources permit #STT-050-10). Samples were collected at depths of 5.5–7.6 m and at temperatures 29 degrees C. Colonies were within 5–7 m of each other and 75 m from shore.

Viromes: Phage and eukaryovirus particles were isolated and sequenced separately from bacterial cells (using CsCl density gradient ultracentrifugation) before sequencing (Vega Thurber et al., 2009; more details in Soffer et al., 2014). DNA was then extracted with a phenol-chloroform extraction protocol (Vega Thurber et al., 2009; Soffer et al., 2014) and amplified using non-specific MDA according to manufacturer’s protocol (GenomPhi, GE Healthcare, Pittsburgh, PA, USA). The coral virome libraries (21 coral samples) were barcoded and pyrosequenced at EnGencore (University of South Carolina) on a Roche Titanium 454 platform. The final numbers of replicate libraries for each coral health state were: H (n = 2), B (n = 5), BD (n = 7) and D (n = 7).

Bacterial Amplicons: 500 ul of ethanol/tissue slurry was pipetted for DNA extractions. A modified organic extraction protocol was used to purify DNA (for details see Soffer, Zaneveld, and Vega Thurber (2015) Phage–bacteria network analysis and its implication for the understanding of coral disease. Environmental Microbiology 17:1203-1218). Isolated nucleic acids were amplified using barcoded primers 515F and 806R, which were chosen due to their ability to amplify both bacteria and archaea (Caporaso et al., 2011; Walters et al., 2011). Triplicate amplicon libraries were prepared using GoTaq Flexi reagents from Promega (Madison, WI, USA) using standard protocols and the following PCR cycle: 1 cycle of 94 degrees C for 3 min, 35 cycles of 94 degrees C for 45 s, 50 degrees C for 60 s, and 72 degrees C for 90 s, and then 1 cycle of 72 degrees C for 10 minutes. PCR products were run on a 1.5 agarose gel, triplicates pooled, cleaned using AMPure magnetic beads from Agencourt (Brea, CA, USA) and quantified with a Qubit HS dsDNA kit (Invitrogen,Eugene, OR, USA) into equimolar ratios. Quality of amplicons were determine on an Agilent Bioanalyser 2100 before being pyrosequenced on a 454 GS Junior Roche at the Oregon State University Center for Genome Research and Biocomputing Core Laboratories.

Related Publications:
Soffer,N., Brandt, M.E., Correa, A.M., Smith, T.B. and Vega Thurber, R.L. 2014. Potential role of viruses in white plague coral disease. ISME J., 8(2): 271-283. doi:10.1038/ismej.2013.137

Soffer, N., Zaneveld, J., and Vega Thurber, R. 2015. Phage–bacteria network analysis and its implication for the understanding of coral disease. Environmental Microbiology, 17:1203-1218. doi:10.1111/1462-2920.12553