302
For each sample, a few mm3 of preserved tissue/skeleton was transferred to 750 l of DNA extraction buffer (100 mM Tris/EDTA/NaCl, 1% SDS) and incubated overnight at 65 C. Proteins were removed from chilled extracts (1 M potassium acetate), and DNA was precipitated (100% isopropanol), washed (70% ethanol), re-suspended (0.1 M Tris), and stored at 4 C. PCR amplifications of the Symbiodinium ITS2 rDNA region were performed following (Arif et al., 2014). The primers ITSintfor2 and ITS2-reverse (LaJeunesse, 2002) were used with adapters (underlined) for sequencing on the Illumina MiSeq platform: 5'- TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGGAATTGCAGAACTCCGTG-3' (MiSeq-ITSintfor2) and 5'-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGGGATCCATATGCTTAAGTTCAGCGGGT-3' (MiSeq-ITS2-reverse). Triplicate 25 l reactions were performed in Qiagen Multiplex Master Mix (Qiagen) and 0.1 M under the following conditions: 94C (5 min); 27 cycles of 94 C (30 sec) 51C (30 sec), 72C (30 sec); 72 C (10 min). Amplification was verified by visualisation on 1% agarose gels. Triplicate PCRs of each sample were pooled and cleaned with the Agencourt AMPure XP magnetic bead system (Beckman Coulter). Nextera XT indexing and sequencing adapters were added via PCR (8 cycles) following the manufacturer's instructions. Sample libraries were quantified on the BioAnalyzer (Agilent Technologies) and QuBit (Quant-IT dsDNA Broad Range Assay Kit; Invitrogen) and pooled in equimolar ratios. This pool of libraries was purified on a 2 % Agarose gel to remove any excess primer and sequenced at 8pM with 10 % phiX on the Illumina MiSeq, 2 x 300bp paired end version 3 chemistry according to manufacturer specifications.