ITS amplicons of coral-associated Symbiodiniaceae
The ITS2 region was amplified using primers ITSintfor2 and ITS2-reverse (LaJeunesse 2002) with Illumina adapters. PCRs were run with 11 l of Multiplex PCR Kit (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 94C, followed by 27 cycles of 94C for 30 s, 51C for 30 s, 72C for 30 s, and a final extension step of 10 min at 72C. PCR products were run on a 1% agarose gel stained with 1x SYBR Safe (Invitrogen, Carlsbad, CA, USA) to visualize successful amplification. The triplicate PCRs were pooled and 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 quantified on an Aligent BioAnalyzer 2100 (Agilent Technologies, Santa Clara, CA, USA) and via qPCR (ThermoFisher Scientific, Waltham, MA, USA) to check library size and concentration, respectively. Libraries were sequenced using the Illumina MiSeq platform (Illumina, San Diego, CA, USA) and kit reagents v3 (2 x 300 bp pair ended reads).