Marine water metagenome
Coral reef water column samples (approximately 5 L) were transported to the laboratory while maintained at 4C. Upon arrival, the water samples were filtered through 0.22 m polycarbonate membrane filters under aseptic conditions. The filters were then immersed in DESS solution and stored at -80C until further processing.Before DNA extraction, the stored membranes were gently rinsed with sterile deionized water and cut into small pieces using sterile scissors. High molecular weight DNA was extracted using the Fast DNA Spin Kit for Soil (MP Biomedicals, USA) following 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.For 16S rRNA gene amplification (V3-V4 region), universal primers 338F and 806R were used. PCR conditions included an initial denaturation at 95C for 2 minutes, followed by 30 cycles of denaturation (95C, 20s), annealing (55C, 30s), extension (72C, 20s), and a final extension at 72C for 5 minutes. The resulting amplicons (~380-400 bp) were confirmed via 2% agarose gel electrophoresis and purified using bead purification.Adaptor ligation was performed through a second PCR using GoldStart Master Mix and UAP1 primers, followed by another round of bead purification. The final library was quantified using Qubit 3.0 and stored at either 4C or -20C. For each water sample, replicate samples underwent library preparation and sequencing.