Dataset: Porites lobata viromes and 16S amplicons
Deployment: Vega_Thurber_Coral_Virus

16S library preparation
Nucleic acid extractions were conducted using the MOBIO UltraClean Microbial DNA Isolation Kit according to the manufacturer’s protocol. Resulting DNA was amplified using barcoded 16S primers (515F/806R) (Caporaso et al., 2011; Walters et al., 2011). Amplicons were amplified in triplicate with GoTaq Flexi reagents from Promega (Madison, WI, USA) using manufacturer's protocols. Triplicate reactions were pooled and cleaned using AMPure magnetic beads from Agencourt (Brea, CA, USA). Quantification of products was conducted with an Invitrogen Qubit HS dsDNA kit (Eugene, OR, USA) and quality was determined on an Agilent Bioanalzer 2100. Equimolar ratios of the amplicons were pooled and sequenced on 454 GS FLX machine at the Center for Genome Research and Biocomputing (CGRB) at OSU.

Virome preparation
Coral tissue cores from the initial sampling (October 2009) were rinsed in viral free seawater then airbrushed into a sterile Tri-cornered beaker. The airbrushed tissue was resuspended in 40 ml of 0.02 um filtered 1X PBS, transferred to a 50 ml sterile conical tube and preserved with 2 ml reagent grade chloroform. All chloroform-persevered samples were stored at 4 degrees C and shipped overnight to the Vega Thurber lab and kept at 4 degrees C upon arrival.

Twenty-four ml of supernatant were filtered through a 1.0 um filter using a swin-lock and 10 ml luer lok syringe to remove large cells and debris. Viral particles were concentrated using CsCl density gradient ultracentrifugation as described (Vega Thurber et al., 2009). Prior to processing all the samples, one sample from each health state (healthy, healthy tissue of GA colonies, and the GA itself) was chosen to determine in which densities the virons were distributed. The presence of viral particles at each density was accessed using SYBR Gold staining (Vega Thurber et al., 2009). Viral particles were primarily distributed within the 1.35 mg ml-1 and 1.2 mg ml-1 densities. The remainder of the samples were then processed and viral particles removed from the 1.35 mg ml-1 and 1.2 mg ml-1 densities in triplicate. Each resulting sample was filtered through a 0.22 um Sterivex filter to remove bacteria. The removal of bacterial cells was confirmed using SYBR Gold staining on several samples.

Viral DNA was extracted from each sample using the protocol outlined by Vega Thurber et al. (2009). To confirm that bacterial and eukaryotic contamination was removed during filtration, we PCR amplified 16S and 18S rDNA in the extracted coral samples. Viral DNA underwent  multiple displacement amplification  in quadruplet using GE Health Sciences Genomphi kit (Pittsburgh, PA, USA). Approximately 2 ug of viral DNA was pyrosequenced at Engencore (University South Carolina) on a Roche 454 Titanium machine. Sequence reads first underwent preprocessing to remove sequences that had low quality scores (less than or equal to 20 average), were short (less than or equal to 100 bases), and/or were duplications using Galaxy (Goecks et al., 2010).