Powerbead tube

To extract DNA, coral tissue (~0.2 cm²) was first placed in a Power Bead Tube (Qiagen) and homogenized horizontally with a vortex adapter for 10 minutes. The samples were then processed using the DNeasy PowerSoil kit (Qiagen, Germantown, MD) following the manufacturer’s protocol. Extracted DNA was PCR amplified with 16S rRNA gene V4 primers^{25 (link)} using the Earth Microbiome Project (EMP) protocols²⁶ . Briefly, samples were processed using the Platinum Hot Start PCR Master Mix (2X) (ThermoFisher Scientific, Waltham, MA) in a 50-µl reaction with 2 µl of DNA template or 2 µl of PCR grade water for the negative control. DNA was amplified with the following parameters: 94 °C for 3 minutes (1X), 94 °C for 45 seconds (35X), 50 °C for 60 seconds (35X), 72 °C for 90 seconds (35X), and 72 °C for 10 minutes (1X). PCR products were cleaned using AMPure XP beads (Beckman Coulter, Brea, CA). Each cleaned, amplified, and barcoded DNA sample was normalized to 4 nM (except for the negative control due to its low DNA quantity). After, 5 µl of each sample and the PCR negative control were all pooled, and sent to the Hussman Institute for Human Genomics University of Miami Miller School of Medicine for sequencing on the MiSeq with PE-300v3 kits.

Stool sample aliqouts—frozen at -80C - were allowed to thaw at room temperature and an aliquot of approx. 250 mg was transferred to a Powerbead tube (Qiagen, Germany). Samples were further processed by addition of 1 mL of ice-cold PBS followed by bead beating in a tissue homogenizer (Bertin, France) for 1 minute at 6,500 rpm. Stool extracts were centrifuged at 23,000 x g for 15 minutes at 4°C and supernatant was transferred to a protein LoBind tube (Eppendorf, Germany) and stored at -20°C till further use. Where indicated the bead beating step was omitted from the procedure.

750μl of nasopharyngeal (NP) swab in viral transport medium was transferred into a Qiagen PowerBead Tube and centrifuged at 13,000g for five minutes at 4°. 500μl of supernatant was transferred into a bioMérieux eMAG (Marcy-l’Étoile, France) for total nucleic acid (TNA) extraction. DNA extraction was performed on the NP swab pellet using DNeasy PowerSoil kit (Qiagen). Nucleic acid extracts were stored at −80°C then thawed on ice at time of analysis. Negative controls (PBS) were processed in parallel to the nasal swab samples to assess contamination during extraction, amplification, and sequencing.

The lysate was pipetted into a 50-mL centrifuge tube. The samples were centrifuged at 2,000 x g for 5 minutes, and the upper colloidal phase was discarded. The middle phase was transferred to a new 50-mL centrifuge tube, and PBS was added to the lysate at a ratio of 1:1. The samples were shaken for approximately 3 minutes, the mixture was centrifuged at 500 x g for 5 minutes, and the supernatant was collected in a sterile 50-mL centrifuge tube. This step was repeated three times. We performed mechanical cell lysis: 1,000 μL of sample was added to a PowerBead tube (Qiagen, Hilden, Germany) and lysed using a MagNA Lyser (Roche Applied Sciences; Penzberg, Germany) at 5,000 RPM for 30 seconds. The samples were centrifuged at 16,000 x g for 1 minute, and the supernatant was pipetted into a sterile Eppendorf tube.

Samples were filtered using 0.2-μm Supor PES membrane disc filters (PALL Life Sciences, Port Washington, NY, United States) using an EZ-Fit 3-place manifold (Merck Millipore, Billerica, MA, United States). After filters were allowed to dry, they were cut with sterile scissors over a sterile 55-mm Petri dish. All the pieces were transferred using sterile tweezers to a PowerBead tube from the DNeasy PowerSoil Kit (Qiagen, Germantown, MD, United States). All these steps were carried out inside a Class II biological safety cabinet. DNA extraction was performed following the manufacturer’s instructions, except for some previously published modifications (Jiang et al., 2015 (link)); and samples were stored in the -80°C freezer until being shipped to the sequencing service.

Isolates had been stored frozen (-80°C) and were recovered on Fastidious Anaerobe Agar (Neogen®, Auchincruive, Scotland) with the addition of 5% horse blood. DNA from 24-hour cultures was manually extracted using the DNeasy UltraClean Microbial Kit (Qiagen, Hilden, Germany), with the following modified pre-step: one-third of a 10 μL loop of cultured bacteria was added to 300 μL PowerBead solution in a PowerBead tube (Qiagen). Thereafter, 50 μL SL solution (part of DNeasy UltraClean Microbial Kit) was added, vortexed briefly and incubated at 95°C for 5 minutes. The tubes were vortexed for 20 minutes and then centrifuged at 10,000×g for 2 minutes and then processed according to the manufacturer’s instructions and eluted in 50 μL.