Samples were collected in two reef systems, the Florida Reef Tract in June of 2019 and off the southern coast of St. Thomas in the U.S. Virgin Islands in Februrary of 2020. See Supplemental File, "sampling_locations.csv" for latitude, longitude, depth, and dates of each sampling location. A total of 3 reef sites at each reef system were sampled. At each reef site, three 10 meter transects were taken by laying down a 10 meter weighted line that was marked every meter. Water samples were taken by a diver using a 60 or 100 mL syringe positioned approximately 5 mm above the benthos at each meter line. The transects were laid haphazardly, but did not intersect with each other. Because of inclement conditions, only 1 transect was collected at Site 73, rather than the intended 3. To capture the seawater microbial community, 60 mL of the seawater was filtered through a 0.22 µm Supor filter (25 mm; Pall Corporation). Filters were placed in 2 mL cryovials, flash frozen in a liquid nitrogen dry shipper, and processed upon return to Woods Hole, MA.
DNA was extracted from the filters using the DNEasy PowerBiofilm Kit (Qiagen) according to manufacturer protocols. Seven DNA extraction controls, consisting of unused 0.22 µm filter, were processed alongside samples. Primers 515F (Parada et al. 2016) and 806R (Apprill et al. 2015) containing Nextera adapter sequences were used to amplify the V4 region of the small subunit rRNA gene in bacteria and archaea.
PCR reactions contained 14.75 µL molecular grade water, 5 µL GoTaq Flexi 5X buffer (Promega Corporation), 2.5 µL of 25 mM MgCl2, 1 µL of 10 mM dNTPs, 1 µL of 10 mM forward and reverse primers, 0.5 µL GoTaq DNA polymerase (Promega). Three PCR controls consisting of 1 µL of PCR-grade water was also included, as well as microbial genomic DNA from a Human Microbiome Project mock community (BEI Resources, NIAID, NIH as part of the Human Microbiome Project: Genomic DNA from Microbial Mock Community B (Even, Low Concentration), v5.1L, for 16S RNA Gene Sequencing, HM-782D). The first stage PCR conditions were: 28 cycles (95°C 20s, 55°C 20s, 72°C 5 min) with a 2 minute 95°C hot start and 10 min 72°C final elongation. PCR products were screened for quality using gel electrophoresis and purified using the MinElute PCR purification kit (Qiagen). PCR products were then barcoded using the Nextera XT Index Kit v2 primers (Illumina) using the following conditions: 8 cycles (95°C 30s, 55°C 30s, 72°C 30s) with 3 minute 95° hot start and 5 minute 72°C final elongation. Barcoded products were purified as above and concentrations of the purified products were assessed using the HS dsDNA assay on the Qubit 2.0 fluorometer (ThermoFisher Scientific). Products were diluted with Tris HCl to 5 nM before being pooled randomly into two libraries. The libraries were diluted to a final loading concentration of 50 pM with a 5% spike-in of 50 pM PhiX. The libraries were then sequenced on the iSeq 100 System (Illumina).
