ITS2 Mhispida Brazil Coral
Amplification of the ITS2 region was completed using the Qiagen Multiplex PCR kit, with 15 ng of DNA from each coral sample using the primers SYM_VAR_5.8S2 [5-GAATTGCAGAACTCCGTGAACC-3] and SYM_VAR_REV [5-CGGGTTCWCTTGTYTGACTTCATGC-3]96 with unique 8-mer barcodes at the respective 5 ends at a final primer concentration of 0.5 M in a reaction volume of 10 l. Thermal cycler conditions for ITS2 PCR amplification were as follows: initial denaturation at 95C for 15 min, 35 cycles at 95C for 30 s, 56C for 90 s, and 72C for 30 s, followed by a final extension step at 72C for 10 min. To confirm successful amplification, 1 l of each PCR product was run on a 1% agarose gel. Samples were cleaned using ExoProStar 1-step (GE Healthcare) and normalized using the SequalPrep Normalization Plate Kit (Thermo Fisher Scientific). The samples were then pooled into a 1.5 mL Eppendorf tube (17 l per sample) and concentrated using a benchtop Speedvac (Concentrator plus, Eppendorf). Quantification was completed using Qubit (Qubit dsDNA High Sensitivity Assay Kit, Invitrogen), and samples were paired-end sequenced (2 x 250bp) on the NovaSeq 6000 platform at the Novogene Sequencing Centre (Cambridge, England).