16S amplicon of oyster microbiomes with nutrient enrichment
Total nucleic acids (TNA) were extracted from the swab samples using the Qiagen Allprep PowerViral DNA/RNA extraction kit with modifications as follows. The swab samples were removed from the RNAlater and each washed with 1 mL of sterile, filtered 1X nuclease-free Phosphate Buffer Saline (PBS, pH 7.4, Invitrogen). The remaining RNAlater was centrifuged to pellet the excess cells, then washed with 1X PBS. The cell pellet was suspended in 600 ?L of Qiagen Solution PV1 and added directly to a 0.1mm glass bead tube, along with the washed swab and 6 ?L of sterile ?-mercaptoethanol. The samples were subjected to bead beating for 5 minutes, followed by proteinase K digestion at 55 C for 1 hour in a shaker at 300 rpm. The supernatant was transferred to a new microcentrifuge tube and the protocol continued according to the manufacturers recommendations. Following TNA extraction, the concentration was quantified using a Nanodrop 2000 instrument (ThermoFisher), and 30 ?L was allocated for DNA. DNA purification of the 30 ?L TNA aliquot was performed using an adapted version of the DNeasy PowerLyzer PowerSoil Kit. In brief, the TNA aliquot was transferred to a new 2 mL microcentrifuge tube and 1200 ?L of Solution C4 was added, then vortexed to mix. Next, 4 ?L of RNase A solution was added to the sample and incubated for 2 minutes at room temperature. The treated DNA was then purified using the spin column and an ethanol wash with Solution C5. The final DNA sample was eluted in 50 ?L of Solution C6. 16S rRNA gene amplicon analysis was performed using 967F/1064R primers to amplify the V6 region.