Cold-water coral microbiomes (Acanthogorgia spp. Desmophyllum dianthus, and Lophelia pertusa) from the Gulf of Mexico and Atlantic Ocean off the southeast coast of the United States: raw sequencing data

Sample collection and preservation: Samples from Norfolk Canyon were collected in 2013 on the National Oceanic and Atmospheric Administration (NOAA) ship Ronald H. Brown using the remotely operated vehicle (ROV) Jason II (research cruise RB-13-03-HBH Deepwater Canyons). The samples acquired from west Florida sites Many Mounds and Okeanos Ridge were collected in 2017 on the NOAA ship Nancy Foster using the ROV Odysseus (NF1708 Southeast Florida Deep Coral Initiative). The 2018 and 2019 collections were part of NOAA's DeepSEARCH program and were conducted on the R/V Atlantis using the human occupied vehicle (HOV) Alvin (AT41) and the NOAA ship Ronald H. Brown using the ROV Jason II (RB1903), respectively. Subsamples of coral colonies were collected using the respective vehicle’s manipulator arm to remove a branch (or in the case of D. dianthus, the entire cup coral). Samples were placed into individual, thermally-insulated containers that had been precleaned (washed with freshwater, interiors wiped with 100% ethanol to remove any biofilms or particulates from prior collections), filled with freshwater, and sent down sealed. When opened to receive a coral collection, the freshwater was replaced by seawater local to the collection site due to density differences. The containers were then sealed at depth to prevent microbial contamination from other sample collections or passing through different water masses during vehicle recovery. At the conclusion of the dive, containers were brought aboard their respective vessel and into a cold room or laboratory and samples were removed using ethanol-sterilized forceps. All corals were lightly rinsed with sterile 1xPBS (phosphate-buffered saline) to remove any loosely associated surface microbes. For octocorals (A. aspera, A. spissa), branches that had not been in contact with the manipulator claw were cut off using ethanol-sterilized shears and transferred to sterile tubes. For the stony corals (D. dianthus, L. pertusa), samples were placed into sterile aluminum weigh boats and a flame-sterilized hammer was used to break open the calyces to expose polyp tissue, which was transferred to sterile tubes. All samples were preserved with RNAlater, stored for 24 hours at 4°C to allow the preservative to infiltrate the tissues, and then moved to -20 °C until processing. Subsamples of the corals collected for microbial analyses were shared with researchers conducting population genetics/taxonomy studies, allowing for accurate host identification of octocorals and the possibility of interpreting microbiome trends against coral genotypes.

DNA extraction: The DNA extraction process was performed on July 30th and July 312st, 2019, at the USGS Coral Microbial Ecology Laboratory. Just prior to extraction, coral samples were rinsed with 0.2 µm-filtered and autoclaved 1xPBS to remove RNAlater. Coral pieces were placed into sterile microcentrifuge tubes using flame-sterilized forceps, 2 ml of the sterile 1xPBS was added to the tube, which was then inverted 3 times and then centrifuged at 4 000 xg for 30 sec. The coral samples were then weighed using sterile technique and approximately 0.1–0.2 g per sample were used for extraction. Extractions were done in duplicate for each coral using Qiagen’s DNeasy PowerBiofilm kits (QIAGEN, 2020; catalog number 24000-50) and replicates were combined at the end. The manufacturer’s protocol was followed with the exception that a FastPrep was used on setting 5 (~3100 rpm) for homogenization in place of a PowerLyzer. Kit blank (extractions with no sample added) were processed at the same time as the samples. DNA was quantified using a Qubit, extractions diluted to approximately 30 ng/µl, and submitted for sequencing.

Sequencing: PCR amplification and sequencing were performed by Michigan State University Genomics Core. The V3-V4 regions of the 16S rRNA gene were amplified with the primers 341F (CCTACGGGAGGCAGCAG) and 806R (GGACTACHVGGGTWTCTAAT). One µl of genomic DNA was added to 7.5 µl of 2x Dream Taq Master Mix and 6.4 µl of a 0.5 µM primer mix. Amplification conditions were an initial melting period of 2 minutes at 95°C, followed by 30 cycles of 95°C for 40 seconds, 50°C for 30 seconds, and 72°C for 60 seconds, and a final anneal step at 72°C for 7 minutes. After PCR the output of all reactions was batch normalized using an Invitrogen SequalPrep DNA Normalization Plate, and all material recovered from the plate was pooled. The pooled material was cleaned up and concentrated using AmpureXP magnetic beads. The pool was quality controlled and quantified using a combination of Qubit dsDNA HS, Agilent 4200 TapeStation HS DNA1000 and Kapa Illumina Library Quantification qPCR assays. This pool was loaded onto an Illumina MiSeq v2 Standard flow cell and sequencing was carried out in a 2x250bp paired end format using a MiSeq v2 500 cycle reagent cartridge. Custom sequencing and index primers complementary to the 341F/806R sequences used for preparing the libraries were added to appropriate wells of the reagent cartridge. Base calling was done by Illumina Real Time Analysis (RTA) v1.18.54 and output of RTA was demultiplexed and converted to FastQ format with Illumina Bcl2fastq v2.19.1.