title: ITS2 sequencing of Millepora platyphylla symbiosis
design: All colony fragments were incubated at 55 C for 1 hour in 450 L of digest buffer with proteinase K (QIAGEN, Hilden, Germany) and DNA was extracted using a QIAxtractor automated genomic DNA extraction instrument, according to manufacturers instructions. The The Symbiodiniaceae ITS2 region of the rDNA was amplified using the primers SYM_VAR_5.8S2 and SYM_VAR_REV (Hume et al. 2018) with added sequencing adapters (forward: 5TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGGAATTGCAGAACTCCGTGAACC3; reverse: 5GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGCGGGTTCWCTTGTYTGACTTCATGC3; Illumina over-hang adaptor sequences are underlined).Ten uL PCRs containing 1 uL of template DNA and 0.25 M of each primer were run in triplicate per sample using the Multiplex PCR Kit (QIAGEN, Hilden, Germany). PCR cycling conditions were 95 C for 15 min, followed by 30 cycles of 95 C for 30 s, 56 C for 90 s, and 72 C for 30 s, with a final extension time of 10 min at 72 C. Amplification success was verified on a 1% agarose gel, and successful triplicate reactions were pooled and cleaned using the illustra ExoProStar PCR and Sequence Reaction Clean-Up Kit (GE Healthcare Life Sciences, Pittsburgh PA, USA). Indexing adaptors were added via PCR (8 cycles) according to the Nextera XT DNA library preparation protocol using the Multiplex PCR Kit. Indexed PCR products were purified and normalized using the SequalPrep Normalization Plate Kit (Invitrogen, Carlsbad CA, USA), subsequently quantified using a Qubit dsDNA HS Kit (Invitrogen, Carlsbad CA, USA), and ran on the Bioanalyzer 2100 (Agilent Technologies, Santa Clara CA, USA) to confirm amplicon length and purity. The  ITS2 gene amplicon library was sequenced at the KAUST BioScience Core Laboratory on the Illumina HiSeq 2500 platform using the rapid-run mode with 2 x 250 bp overlapping paired-end reads with a 10% phiX control.