title: Orbicella faveolata prokaryome (16S rRNA): Mid-bleached 2 control replicate 2
design: The Symbiodiniaceae communities in the original coral colony, the experimental cores, and the expelled symbionts were assessed using qPCR (Correa, A. M. S., McDonald, M. D. & Baker, A. C., 2009).The remaining coral tissue and skeleton after dissociation was divided into triplicates and DNA was extracted using the ZymoBIOMICS DNA Miniprep Kit (Zymo Research, Irvine, California, USA). The V4 region of the prokaryotic 16S rRNA gene was amplified according to the Earth Microbiome Project protocol (Thompson, L. R. et al., 2017)  and the V4 region of the eukaryotic 18S rRNA gene was amplified using a validated nested PCR approach (del Campo, J. et al., 2019)  which first amplified the DNA with UNonMet-PCR primers to reduce the metazoan signal (18S-EUK581-F 5-GTGCCAGCAGCCGCG-3_, 18S-EUK1134-R 5-TTTAAGTTTCAGCCTTGCG-3_), then reamplified with general eukaryotic primers (18S-E572F 5-CYGCGGTAATTCCAGCTC-3, 18S-E1009R 5- AYGGTATCTRATCRTCTTYG-3) (Comeau, A. M., Li, W. K. W., Tremblay, J. ., Carmack, E. C. & Lovejoy, C., 2011). DNA concentrations from all samples were checked using a Qubit fluorometer before being sent to Novogene (Sacramento, California, USA) for further sequencing. After sequencing, primers were removed from reads using Cutadapt v3.1 (Martin, M., 2011). The trimmed reads were processed in R using DADA2 (Callahan, B. J. et al., 2016). For the 18S rRNA gene amplicons, both the forward and reverse reads were truncated at 210 bp. Reads were then denoised and merged into amplicon sequence variants (ASVs) before removing chimeras using the removeBimeraDenovo command with the consensus option. Taxonomy was assigned to 18S ASVs in DADA2 using PR2 v4.13.0 (Guillou, L. et al., 2013). For the 16S rRNA gene amplicons, both the forward and reverse reads were truncated at 165 bp before denoising and merging into ASVs. Taxonomy was assigned to the 16S ASVs using SILVA v138 (Quast, C. et al., 2012) in DADA2. Sequence tables, taxonomy tables, and metadata for both the 16S and 18S rRNA gene amplicon datasets were uploaded into a Phyloseq object in R for further filtering and analysis (McMurdie, P. J. & Holmes, S., 2013). For the 16S rRNA gene amplicons, ASVs corresponding to chloroplast and mitochondria were removed. For the 18S rRNA gene amplicons, ASVs corresponding to Metazoa and Embryophyceae were filtered from the dataset. For both metabarcoding datasets, ASVs were kept for further analysis only if they were in the most abundant 99% of taxa in at least one of the samples. For the 16S rRNA gene sequencing, 4,966,375 paired-end reads were generated, resulting in 3,756 ASVs across 24 samples (triplicate samples across the four stages of bleaching) following filtering. For the 18S rRNA gene sequencing, 4,701,974 paired-end reads were generated, resulting in 1,419 ASVs across 24 samples following filtering. Bubble plots and diversity figures were constructed using ggplot2 and tidyverse packages (Wicham, H. et al., 2019). Analysis of Compositions of Microbiomes with Bias Correction (ANCOM-BC) (Lin, H. & Peddada, S. D., 2020) was used to find significantly differentially abundant genera between bleaching time points. For the ANCOM-BC test, only genera above 1% relative abundance in each respective dataset were preserved, an alpha of 0.005 was used, and p-values were adjusted using false discover rate (fdr). To place the Symbiodiniaceae ASVs in a phylogenetic context, the top 10 ASVs corresponding to Breviolum and Durusdinium were combined with a Polarella glacialis (GQ375263) outgroup, aligned using MAFFT, then a maximum-likelihood phylogenetic tree was constructed using RAxML (GTRCAT substitution model, N = 1,000) (Katoh, K. & Standley, D. M., 2013; Stamatakis, A., 2014). The resulting tree was modified for publication using ggtree (Yu, G., Smith, D. K., Zhu, H., Guan, Y. & Lam, T. T. Y., 2017).