Coral microbiome preservation and extraction method comparison of samples collected in March and August 2018-raw data

All tropical coral samples (Montastraea cavernosa, Porites astreoides, and Stephanocoenia intersepta) were collected manually by permit in March 2018 from a nursery in the Florida Keys National Marine Sanctuary, temporarily housed at the facility of Mote Marine Laboratory & Aquarium’s Elizabeth Moore International Center for Coral Reef Research & Restoration in Summerland Key, Florida. Samples beginning with MCR, PAR, and SIR were transferred to sterile tubes, covered in RNAlater, and incubated overnight at 4°C to allow the preservative to permeate the coral tissues before transfer to -20°C for long-term storage. Samples beginning with MCS, PAS, and SIS were transferred to sterile tubes, covered in DNA/RNA Shield, and incubated overnight at 4°C to allow the preservative to permeate the coral tissues before transfer to -20°C for long-term storage. Samples beginning with MCN, PAN, and SIN were double-bagged in sterile Whirl-Pak bags (Nasco, Fort Atkinson, WI, USA) and then stored in liquid nitrogen until transfer to -80°C for long-term storage.

All cold-water coral samples (Lophelia pertusa and Paragorgia johnsoni) were collected by the sampling arm of a crewed submersible. The sampler’s individual compartments were cleaned at the surface using ethanol, filled with sterile deionized water, and sealed. Coral branches were collected and placed into the containers after ambient seawater evacuated the freshwater, and the containers were re-sealed at depth. Samples with names beginning with LP and PJ were collected in August 2018 on cruise AT-41 on the R/V Atlantis using the Alvin submersible (Woods Hole Oceanographic Institution). Samples with names beginning with LP were collected from Richardson Hills on dive A4963, while samples with names beginning with PJ were collected from Pamlico Canyon on dive A4969. Upon return to the surface, samples beginning with LPR, PJR, LPS, and PJS were transferred to sterile tubes, covered in preservative, and incubated overnight at 4°C to allow the preservative to permeate the coral tissues before transfer to -20°C for long-term storage. The preservative used for samples LPR and PJR was RNAlater (Life Technologies, Grand Island, NY, USA), while the preservative used for samples LPS and PJS was DNA/RNA Shield (Zymo Research, Irvine, CA, USA). Samples beginning with LPN and PJN were transferred to sterile tubes and then stored in liquid nitrogen until transfer to -80°C for long-term storage.

Homogenization of cells and extraction of DNA from the samples occurred at the Coral Microbial Ecology Laboratory in St. Petersburg, FL. All samples were wrapped in sterile aluminum foil and crushed with a hammer. For samples ending in numbers 1, 2, and 3, cells were disrupted by adding the crushed samples and 300 microliters of molecular grade water to Lysing Matrix A and homogenizing them for 20 seconds in a FastPrep bead mill homogenizer at setting 6.5 (MP Biomedicals, Santa Ana, CA). DNA was extracted from those samples using the Maxwell RSC Blood DNA Kit with a Maxwell RSC Instrument (model AS4500) (Promega, Fitchburg, WI). DNA Lysis Buffer (300 microliters) and Proteinase K (25 microliters) from the kit were added to each sample. Samples were mixed by inversion three times, incubated for one hour in a 56°C, mixed again by inversion three times, and centrifuged at 2500 rpm for 30 seconds. The supernatant of each sample was transferred to a front well of the cartridge in the Maxwell RSC Instrument. Sections 3B and 5 of the manufacturer’s protocol (Promega, 2018) were then followed to complete the DNA extraction. DNA was extracted from samples ending in numbers 4, 5, and 6 using the Qiagen PowerBiofilm DNA Isolation Kit according to the manufacturer’s instructions (Qiagen, 2015) with two modifications to adapt the protocol to the samples. Bead beating (Step 5 of the homogenization section of the protocol) was accomplished using a FastPrep homogenizer for 30 seconds at setting 5. Step 3 of the vacuum section of the protocol was conducted by centrifuging the samples for 1 minute at 13000xg. Two extraction blanks for each method were also prepared: one substituted phosphate-buffered saline solution for the sample and the other substituted nuclease-free water for the sample.

Library preparation and sequencing were conducted by Glomics, Inc. (Norman, OK). To target the V4 region of the 16S rRNA gene, a fusion primer set was constructed using primers 515F (5′ GTGCCAGCMGCCGCGGTAA) and 806RB (5′ GGACTACNVGGGTWTCTAAT) (Apprill and others, 2015) along with adapters, indices, linkers, and pads in accordance with the dual-index sequencing strategy of Kozich and others (2013). Amplicons were purified, quantified, and pooled in equal concentrations for sequencing on a MiSeq sequencing system with v2 chemistry to obtain paired-end 250-bp reads. A mock community was also sequenced in order to assess any biases in the sequencing technology. The raw data files associated with this data release have also been submitted to the NCBI Sequence Read Archive (SRA) under Bioproject number PRJNA544686.