ITS amplicons of coral-associated Symbiodiniaceae
The ITS2 region was amplified using primers SYM_VAR_5.8S2 and SYM_VAR_REV (Hume et al 2018) with Illumina adapters. PCRs were run with 11 l of Multiplex PCR Master Mix (Qiagen, Hilden, Germany), 2 l of each primer (10 M), 5 l DNA, in a total volume of 25 l. The following PCR conditions were used: 15 min at 95C, followed by 30 cycles of 95C for 30 s, 56C for 30 s, 72C for 30 s, and a final extension step of 10 min at 72C. PCR products were checked using QIAxcel DNA Screening Kit (Qiagen) with QIAxcel Advanced System (Qiagen) to visualize successful amplification. PCRs were cleaned with Agencourt AMPure XP magnetic bead system (Beckman Coulter, Brea, CA, USA). Illumina Nextera XT indexing and sequencing adapters were added via PCR (8 cycles) following the manufacturers instructions. Samples were normalized and pooled using SequalPrep Normalization Plate Kit (ThermoFisher Scientific, Waltham, MA, USA). The samples were then analysed on 4200 TapeStation system (Agilent, Santa Clara, CA, USA) and QuantStudio 3 real-time PCR system (Thermo Fisher Scientific) to check library size and concentration, respectively. Libraries were sequenced using the Illumina MiSeq platform (Illumina, San Diego, CA, USA) and MiSeq Reagent Kit v3 (2 x 300 bp paired-end reads).