[Adapted from: Randall et al. 2016 PLoS ONE 11(1): e0147493. doi:10.1371/journal.pone.0147493]
Immediately following completion of the waterborne-transmission experiments (See Randall et al. 2016 PLoS ONE 11(1) attached), three each, of diseased, exposed, and healthy colonies of S. siderea were randomly selected for bacterial-community analyses, to determine whether potential bacterial pathogens had transmitted to the exposed colonies. The nine coral colonies were placed in individual, sterile whirl-paks at -80 degrees C and then were transported on dry ice to Mote Marine Laboratory in Sarasota, Florida.
Tissue was removed from the skeleton of the preserved-coral colonies using a Paasche airbrush with 10 mL of sterile seawater. The tissue slurry was collected in a sterile 50 mL Falcon® tube and homogenized using a vortex. The tissue homogenate was then spun down into a pellet using a centrifuge set at 10,000 rpm. The pellet was re-suspended in 2 mL of solution C1 and DNA was extracted using a Powersoil DNA extraction kit (MoBIO Laboratories Inc. Lot #PS14F19). Extracted DNA was then sent to MRDNA Laboratory (www.mrdnalab.com, Shallowater, TX, USA) for Illumina sequencing (20,000 reads per assay) using the universal bacterial primers 27F/519R with a barcode on the forward primer. The 16S rRNA gene on the V1 – V3 hypervariable region was amplified by applying a 30 cycle polymerase chain reaction (PCR) with the HotStarTaq Plus Master Mix Kit (Qiagen, USA). PCR was applied using the following protocol: (1) 94 degrees C for 3 minutes, (2) 28 cycles of: 94 degrees C for 30 seconds, 53 degrees C for 40 seconds, and 72 degrees C for 1 minute, and (3) a final elongation step at 72 degrees C for 5 minutes. After amplification, PCR products were confirmed in 2% agarose gels to determine the success of amplification and the relative intensity of the bands. Multiple samples were pooled together in equal proportions based on their molecular weight and DNA concentrations. Pooled samples were purified using calibrated Ampure XP beads. Then the pooled and purified PCR product was used to prepare DNA libraries by following the Illumina TruSeq DNA library preparation protocol. Sequencing was performed using the Illumina sequencing platform at MR DNA (www.mrdnalab.com, Shallowater, TX, USA) following the manufacturer’s guidelines. Sequence data were processed using a standardized analysis pipeline. Briefly, sequences were initially depleted of barcodes. Then sequences less than 150bp or with ambiguous base calls were removed. Operational taxonomic units (OTUs) were generated, and chimeras were removed using UCHIME [48]. OTUs were defined by clustering at 3% divergence (i.e., showing 97% similarity) using a de novo method. Final OTUs were taxonomically classified using BLASTn against the curated National Center for Biotechnology Information (NCBI) database and the Ribosomal Database Project (RDP).
Field collection:
Wonderland Reef, Florida (24.56028 N, 81.50127 W). Collections in July 2013.
Laboratory experimentation:
Mote Marine Laboratory, Tropical Research Laboratory, Summerland Key, Florida from 10 July – 14 August 2013.
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