CBASSvsCLASSIC: Symbiodiniaceae ITS2 of Stylophora pistillata central Red Sea
DNA isolation was performed using the Qiagen DNeasy 96 Blood & Tissue kit (Qiagen, Hilden, Germany) following the manufacturer's instructions with minor adjustments. Briefly, the coral tissue samples aliquoted for DNA isolation (see above) were thawed and equilibrated to room temperature. The slurry was vortexed and 180 l of each sample was transferred to a microtube with 20 l of proteinase K for incubation at 56C for 1 hour. DNA extractions were then continued according to manufacturers instructions. DNA concentrations were quantified by Qubit (Qubit dsDNA High Sensitivity Assay Kit, Invitrogen, Carlsbad, USA). ITS2 (Symbiodiniaceae) amplicon libraries were prepared for sequencing on the Illumina MiSeq platform. To amplify the ITS2 region, the primers SYM_VAR_5.8S2 [5-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGGAATTGCAGAACTCCGTGAACC-3] and SYM_VAR_REV [5-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGCGGGTTCWCTTGTYTGACTTCATGC-3] (Hume et al., 2013, 2015, 2018) were used (Illumina adaptor overhangs underlined). Triplicate PCRs were performed (10-50 ng of DNA from each coral sample) with the Qiagen Multiplex PCR kit, with a final primer concentration of 0.5 M in a final reaction volume of 10 l. Thermal cycler conditions for ITS2 PCR amplification were: initial denaturation at 95C for 15 min, 30 cycles of 95C for 30 s, 56C for 90 s, and 72C for 30 s, followed by a final extension step of 72C at 10 min. Then, 5 l of the PCR products were ran on a 1% agarose gel to confirm successful amplification. Triplicates for each sample were pooled, and samples were cleaned using ExoProStar 1-step (GE Healthcare, Chicago, IL, UK). Samples were then indexed using the Nextera XT Index Kit v2 (dual indexes and Illumina sequencing adaptors added). Successful addition of indexes was confirmed by comparing the length of the initial PCR product to the corresponding indexed sample on a 1% agarose gel. Samples were then cleaned and normalized using the SequalPrep Normalization Plate Kit (Invitrogen, Carlsbad, USA). The ITS2 libraries were pooled in an Eppendorf tube (4 l per sample) and concentrated using a CentriVap Benchtop Vacuum Concentrator (Labconco, Kansas City, MO, USA). Following this, quality of the library was assessed using the Agilent High Sensitivity DNA Kit on the Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, USA). Quantification was done using Qubit (Qubit dsDNA High Sensitivity Assay Kit, Invitrogen, Carlsbad, USA). Sequencing was performed at 6 pM with 20% phiX on the Illumina MiSeq platform at 2 x 301 bp paired-end V3 chemistry according to the manufacturers specifications