title: 39-New Cal 16S
design: Fragments (n= 2-5) were sampled for each of three key coral species found throughout the mangrove lagoon and reef sites; A. muricata, A. pulchra, P. lutea . Each replicate sample was taken from independent colonies. Small subsamples were taken from each fragment for Symbiodinium cell density assessment using a haemocytometer and normalized to surface area. Fragments were then preserved in RNAlater for symbiont ITS2 genotyping. Coral fragments were thawed slowly on ice and removed from the RNAlater solution using sterile forceps and kimwipes to remove excess solution. Coral fragments were transferred into sterile zip-lock bags and subsequently air-blasted using airflow from a sterile pipette tip (1000 L filter barrier tips, Neptune, USA) into 5 ml PBS-EDTA with care was taken to exclude any skeletal fragments. DNA was extracted from the tissue slurry using the Qiagen DNeasy Plant Mini Kit (Qiagen, Hilden, Germany) according to manufacturer's instructions. To disrupt Symbiodinium cells, 200 l tissue slurry in 200 l AP-1, 4 l RNase A stock solution (100 g/ml), and 200 l 0.5 mm sterile glass beads (BioSpec, Bartlesville, OK) were bead-beaten at 30 Hz for 90 s with a Tissue Lyser II (Qiagen, Hilden, Germany). An additional step using phenol:chloroform:isoamyl alcohol (25:24:1) pre-buffered to pH 8 was used to purify the DNA. Extracted DNA was quantified and quality checked using a NanoDrop 2000C spectrophotometer (Thermo Fisher Scientific, Waltham, MA). PCR amplifications were performed in triplicate reactions with Qiagen Multiplex PCR Kit (Qiagen, Hilden, Germany). To amplify the bacterial 16S rRNA gene, we used the primers 16SMiSeqF-Andersson 5-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGAGGATTAGATACCCTGGTA-3 and 16SMiSeqR-Andersson 5-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGCRRCACGAGCTGACGAC-3 (Illumina sequencing adapters underlined) that target the variable regions 5 and 6 of the 16S gene. These primers have previously been reported to amplify well with coral DNA. The thermal conditions for 16S amplicon PCRs were as follows: 95C for 15 min, followed by 27 cycles of 95C for 40 s, 55C for 40 s, 72C for 40 s, and a final extension cycle of 72C at 10 min. Sample triplicates were subsequently pooled and then purified using the Agencourt AMPure XP magnetic bead system (Beckman Coulter, Brea, CA, USA). Purified PCR products were subjected to an indexing PCR (8 cycles) to add Nextera XT indexing and sequencing adapters (Illumina) according to the manufacturer's protocol. Indexed amplicons were again purified, quantified on the QuBit (Quant-IT dsDNA Broad Range Assay Kit; Invitrogen, Carlsbad, CA, USA) and pooled in equimolar ratios on the BioAnalyzer (Agilent Technologies, Santa Clara, CA, USA). The final pooled library was purified on a 2% agarose gel to remove excess primer dimer. The library was sequenced at 8pM with 10% phiX on the Illumina Miseq, 2x 300 bp end version 3 chemistry according to the manufacturer's specifications at the Bioscience Core lab (KAUST, Saudi Arabia).