Raw sequencing and amplicon sequence variant data from bacterial communities shed by Montastraea cavernosa coral fragments into filtered seawater mesocosms

Coral collection - The coral fragment for mesocosm McH-101 was collected in April 2018 from the Florida Keys while the fragment for mesocosm McH-103 was acquired on January 31, 2019, from the Florida Keys National Marine Sanctuary in Key West. Both healthy coral fragments were maintained in a holding tank at the Smithsonian Marine Station located in Ft. Pierce, FL, until the first mesocosm experiment began in October 2019. Coral samples for mesocosms Mcav17 and Mcav18 were collected from a patch reef near Summerland Key (24.54767, -81.45697) on October 23, 2019. Coral samples for mesocosms McD-57 and McD-58 were obtained from a patch reef near Marathon (24.68982, -81.02978) on November 1, 2020. The second mesocosms were inoculated at the Smithsonian Marine Station in November 2020.

Sample creation - first mesocosm inoculations: The mesocosms were initially set up and processed at the Smithsonian Marine Station (27.45956, -80.31060). Mesocosm seawater came from a storage tank that recirculated it through an ultraviolet (UV) sterilizer, 20-micrometer (µm) filter, and an activated carbon filter. The seawater was 0.22-µm filtered prior to use in 18-liter (L) mesocosms and is referred to as ‘filtered seawater.’ For samples McH-101, McH-103, Mcav17, and Mcav18, coral fragments were placed in filtered seawater mesocosms and allowed to naturally shed microbes from October 25 to October 29, 2019.

Sample creation - second mesocosm inoculations: The second mesocosms were also instituted and processed at the Smithsonian Marine Station in Ft. Pierce, Florida, using the same configurations described in the previous step. For sample McD-57, the coral fragment was placed in filtered seawater mesocosm and allowed to naturally shed microbes from November 3-6, 2020. For sample McD-58, the coral fragment was placed in filtered seawater mesocosm and allowed to naturally shed microbes from November 3 to November 7, 2020.

Sample creation - first filtration: Samples McH-101 and McH-103 were processed on October 28, 2019, and samples Mcav17 and Mcav18 were processed October 29, 2019. For samples McH-101, McH-103, Mcav17, and Mcav18, the coral fragments were removed from the 18L mesocosms and the water was pre-filtered through a sterile 200-micron mesh to remove loose debris. A Pall tangential flow filtration (TFF) system employing five 100 kDa T-series Centramate filter cassettes was used to concentrate the microbial community in each sample from the 18L original volume down to 210-250 milliliter (ml) concentrate. The microbial concentrate was filtered through a sterile Corning 430756 unit containing a 0.22-µm cellulose nitrate filter and then the filter was cut out of the unit with a sterile razor blade, transferred to a sterile whirlpak bag and frozen at -20°C until extracted for DNA.

Sample creation - second filtration: Sample McD-57 was processed November 6, 2020. Sample McD-58 was processed November 7, 2020. For samples McD-57 and McD-58, the coral fragments were removed from the 18L mesocosms and the water was pre-filtered through a sterile 106-micron mesh to remove loose debris. A Pall tangential flow filtration system employing five 100 kDa T-Series Centramate filter cassettes was used to concentrate the microbial community in each sample from the 18L original volume down to 190-230 ml concentrate. The microbial concentrate was first filtered through a sterile ThermoScientific 126-0080 unit containing a 0.8-µm cellulose nitrate filter to remove any eukaryotes. The filtrate was then filtered through a sterile Corning 430756 unit containing a 0.22-µm cellulose nitrate filter and then the filter was cut out of the unit with a sterile razor blade, transferred to a sterile whirlpak bag and frozen at -20°C, until extracted for DNA.

DNA extraction - The DNA extraction process was performed in March 2021, at the USGS Coral Microbial Ecology Laboratory located in St. Petersburg, FL. DNA was extracted from the 0.22-µm cellulose nitrate filters using Qiagen’s DNeasy PowerBiofilm Kit (QIAGEN, 2020), following manufacturer’s instructions in the Quick Start Protocol dated November 2016.

Sequencing - Sequencing was done by the Michigan State University RTSF Genomics Core (East Lansing, MI). USGS submitted eight samples (n=6, plus one mock community and one extraction kit blank) of microbial metagenomic DNA for amplicon library preparation of the bacterial 16S-V4 hypervariable region and Illumina sequencing. Libraries were prepared using the MiSeq Dual Index method developed in the Patrick Schloss lab (Kozich and others, 2013). Primers 515f/806r were used to amplify the V4 region. All reactions were batch normalized using Invitrogen SequalPrep DNA Normalization plates. Product recovered from the plates was pooled and concentrated using an Amicon spin column. This pool was cleaned up using AMPure XP magnetic SPRI 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. The pool was loaded onto an Illumina MiSeq v2 Nano flow cell and sequencing was performed in a 2x250bp paired end format using a v2 500 cycle reagent cartridge. Custom sequencing and index primers complementary to the 515f/806r oligomers 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 v2.20.0.

Data analysis - Data files (BacteriaFilters_ASV_Table.zip) containing tabulations of ASVs, their DNA sequences, their taxonomic identification, and the abundance (number of sequence reads) for each of the samples and controls were added in the version 2.0 update of this data release (Kellogg and others, 2021). Sequences were imported into QIIME2 [version 2021.4] (Bolyen and others, 2019) and trimmed, merged, and sorted into amplicon sequence variants (ASVs) using DADA2 (Callahan and others, 2016) and the parameters ‘–p-trim-left-f 13 –p-trim-left-r 13 –p-trunc-len- f 200 –p-trunc-len-r 200.’ Taxonomy was assigned using the pre-trained Silva classifier silva-138-99-515-806-nb-classifier (Bokulich and others, 2018). All ASVs assigned as “mitochondria” or “chloroplast” were removed.