title: Favia_triplicate _03
design: Coral samples preserved in DESS solution were washed with sterile deionized water to remove salt and debris. Coral tissues were extracted under aseptic conditions using an air compressor and delivery gun, aliquoted, and stored at -80C until processing. For DNA isolation, samples were thawed at 4C and extracted using the Fast DNA Spin Kit (MP Biomedicals, USA) per the manufacturers protocol. DNA quality was assessed via agarose gel electrophoresis, NanoDrop ND-2000 (Thermo Fisher, USA) for purity, and Qubit 3.0 Fluorometer (Invitrogen, USA) for quantification. 16S rRNA gene amplification (V3-V4 region) was performed using universal primers 338F and 806R. PCR conditions: initial denaturation (95C, 2 min), 30 cycles of denaturation (95C, 20s), annealing (55C, 30s), extension (72C, 20s), and final extension (72C, 5 min). Amplicons (~380-400 bp) were confirmed by 2% agarose gel electrophoresis and purified using bead purification. For adaptor ligation, a second PCR was performed with GoldStart Master Mix and UAP1 primers, followed by bead purification. The final library was quantified using Qubit 3.0 and stored at 4C or -20C.