Amplicon sequencing of Pseudodiploria strigosa tissue microbiome
For DNA extraction, up to 0.040 g of the coral tissue pellet was weighed and processed using the DNeasy PowerBiofilm Kit (Qiagen, Inc., Germantown, MD) following the manufacturers instructions. A blank extraction was included alongside the samples. The extracted DNA served as the template for 16S rRNA gene sequencing to identify the microbiome taxonomically. Single, barcoded PCR reactions were performed to amplify the bacterial and archaeal small subunit ribosomal RNA (rRNA) gene using primers 515FY and 806RB (Apprill et al., 2015). As a sequencing control, we used genomic DNA from Microbial Mock Community B (even, low concentration), v5.1L, HM-782D, and nuclease-free PCR-grade water was used as a negative control. Each 50 L PCR reaction contained: 2 L DNA template, 0.5 L GoTaq DNA Polymerase (Promega), 1 L of each forward and reverse primer at 10 M, 1 L of 10 mM deoxynucleoside triphosphate (dNTP) mix (Promega), 5 L MgCl, 10 L GoTaq 5X colourless flexi buffer (Promega), and 29.5 L nuclease-free water. The PCR cycling conditions were as follows: initial denaturation at 95C for 2 minutes; followed by 34 or 37 cycles of 95C for 20 seconds, 55C for 15 seconds, and 72C for 5 minutes; and a final extension at 72C for 10 minutes, before holding at 4C. We used both 34 and 37 cycles for samples to obtain comparable amounts of amplified DNA for subsequent purification and sequencing. Barcoded PCR products were purified using the MinElute PCR Purification Kit (Qiagen, Inc., Germantown, MD), and concentrations were measured using a Qubit 2.0 fluorometer. Purified samples were diluted to 1 ng/L and pooled for sequencing. Sequencing was performed with 2x250 bp paired-end reads on an Illumina MiSeq platform at the University of Illinois at Urbana-Champaign. The resulting fastq files were then downloaded and analyzed.