From Baumann et al (2017):

DNA Extraction

Coral holobiont (coral, algae, and microbiome) DNA was isolated from each sample following a modified phenol-chloroform [86,87,88] method described in detail by Davies et al. [87]. Briefly, DNA was isolated by immersing the tissue in digest buffer (100 mM NaCL, 10 mM Tris-Cl pH 8.0, 25 mM EDTA pH 9.0, 0.5% SDS, 0.1 mg ml-1 Proteinase K, and 1 µg ml-1 RNaseA) for 1 h at 42 °C followed by a standard phenol-chloroform extraction. Extracted DNA was confirmed on an agarose gel and quantified using a Nanodrop 2000 Spectrophotometer (Thermo Scientific).

PCR Amplification and Metabarcoding

The ITS-2 region (350 bp) was targeted and amplified in each sample using custom primers that incorporated Symbiodinium specific ITS-2-dino-forward and its2rev2-reverse regions [65, 73, 89]. Each primer was constructed with a universal linker, which allowed for the downstream incorporation of Illumina specific adapters and barcodes during the second PCR as well as four degenerative bases whose function was to increase the complexity of library composition. The forward primer was 5'-GTCTCGTCGGCTCGG + AGATGTGTATAAGAGACAG+ NNNN + CCTCCGCTTACTTATATGCTT-3', where the underlined bases are the 5'-universal linker, italicized bases indicate spacer sequences, Ns denote degenerative bases, and the bold bases are the ITS-2-dino. The reverse primer was 5'-TCGTCGGCAGCGTCA + AGATGTGTATAAGAGACAG + NNNN + GTGAATTGCAGAACTCGTG-3'.

Each 20 µL PCR reaction contained 5-100 ng DNA template, 12.4 µL Milli-Q H2O, 0.2 µM dNTPs, 1 µM forward and 1 µM reverse primers, 1× Extaq buffer, and 0.5 U (units) Extaqpolymerase (Takara Biotechnology). PCR cycles were run for all samples using the following PCR profile: 95 °C for 5 min, 95 °C for 40 s, 59 °C for 2 min, 72 °C for 1 min per cycle and a final elongation step of 72 °C for 7 min. The optimal number of PCR cycles for each sample was determined from visualization of a faint band on a 2% agarose gel (usually between 22 and 28 cycles) as per Quigley et al. [65]. PCR products were cleaned using GeneJET PCR purification kits (Fermentas Life Sciences), and then a second PCR reaction was performed to incorporate custom barcode-primer sequences [65] modified for Illumina Miseq as in Klepac et al. [90]. Custom barcode primer sequences included 5'-Illumina adaptor + 6 bp barcode sequence + one of two universal linkers-3' (e.g., 5'-CAAGCAGAAGACGGCATACGAGAT + GTATAG + GTCTCGTGGGCTCGG-3', or 5'-AATGATACGGCGACCACCGAGATCTACAC + AGTCAA + TCGTCGGCAGCGTC-3'). Following barcoding, PCR samples were visualized on a 2% agarose gel and pooled based on band intensity (to ensure equal contributions of each sample in the pool). The resulting pool was run on a 1% SYBR Green (Invitrogen) stained gel for 60 min at 90 V and 120 mA. The target band was excised, soaked in 30 µL of Milli-Q water overnight at 4 °C, and the supernatant was submitted for sequencing to the University of North Carolina at Chapel Hill High Throughput Sequencing Facility across two lanes of Illumina MiSeq (one 2 × 250, one 2 × 300). The two lanes produced similar mapping efficiencies (73 and 73%, respectively; Table S3).

Bioinformatic Pipeline

The bioinformatic pipeline used here builds upon previous work by Quigley et al. [65] and Green et al. [73]. Raw sequences were renamed to retain sample information, and then all forward (R1) and reverse (R2) sequences were concatenated into two files, which were processed using CD-HIT-OTU [91]. CD-HIT-OTU clusters concatenated reads into identical groups at 100% similarity for identification of operational taxonomic units (OTUs). Each sample was then mapped back to the resulting reference OTUs, and an abundance count for each sample across all OTUs was produced. A BLASTn search of each reference OTU was then run against the GenBank (NCBI) nucleotide reference collection using the representative sequence from each OTU to identify which Symbiodinium lineage was represented by each OTU (Table S2).

The phylogeny of representative sequences of each distinct Symbiodinium OTU was constructed using the PhyML tool [92, 93] within Geneious version 10.0.5 (http://geneious.com) [94]. PhyML was run using the GTR+I model (chosen based on delta AIC values produced from jModelTest [92, 95]) to determine the maximum likelihood tree. The TreeDyn tool in Phylogeny.fr was used to view the tree (Fig. 2) [96, 97, 98]. The reference sequences included in the phylogeny were accessed from GenBank (Table S6).

These data are reported in:
Baumann, J.H., Davies, S.W., Aichelman, H.E. and Castillo, K. D. (2017) Coral Symbiodinium Community Composition Across the Belize Mesoamerican Barrier Reef System is Influenced by Host Species and Thermal Variability. Microb Ecol. https://doi.org/10.1007/s00248-017-1096-6.

Methodology References:

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