Dataset: Coral ESVs: Sargassum

Methodology:

Acropora millepora corals were subjected to one of nine experimental treatments for 33 days: (1) direct contact with four thalli of Galaxaura rugosa (live seaweed), (2) close proximity (i.e. ~1.5cm away, no contact) to four thalli of Galaxaura, (3) direct contact with four Galaxaura mimics (microfiber dust cloth), (4) close proximity to four Galaxaura mimics, (5) direct contact with four Sargassum polycystum thalli (live seaweed), (6) close proximity to four Sargassum thalli, (7) direct contact with four Sargassum mimics (plastic aquarium plants), (8) close proximity to four Sargassum mimics, or (9) no seaweed or mimic exposure (control) (n = 9-13 per treatment). Analyses of microbiome data were conducted separately to compare control corals and corals in direct contact or close proximity with Galaxaura or its mimics or Sargassum or its mimics. 

Sampling and analytical procedures: 

Total DNA was extracted from each coral sample by placing the clipping directly into a PowerBead tube from the PowerSoil DNA isolation kit (MO BIO Laboratories) and proceeding according to the manufacturer’s instructions. The V3-V4 variable region of the 16S rRNA gene was amplified from 1 µl (25 µl total reaction volume) using the Platinum PCR SuperMix (Life Technologies) and the universal 16S rRNA gene primers 515F (Parada) (5'-GTGYCAGCMGCCGCGGTAA-3') and 806R (Apprill) (5'-GGACTACNVGGGTWTCTAAT-3'). Primers were modified with sample-specific barcodes and Illumina sequencing adapters according to Kozich et al. (2013) to allow for multiplexing of samples. Primers were added to each PCR reaction at a final concentration of 0.2 µM and PCR cycling conditions were: initial denaturation at 94°C for 3 min, followed by 30 cycles of denaturation for 45 s (94°C), primer annealing for 60 s (55°C), extension for 90 s (72°C), and a final 10 min extension step (72°C). PCR products were run on a 1% agarose/1X TAE gel to verify amplicon size and lack of contamination. PCR products were purified using Diffinity RapidTips (Sigma Aldrich) and quantified using the Qubit 2.0 fluorometer. Equimolar concentrations of all samples were pooled and sequenced on a MiSeq sequencer using a 500-cycle paired-end MiSeq Reagent V2 Kit (Illumina).

After sequencing and de-multiplexing, barcoded sequences were trimmed and filtered using Trim Galore! (http://www.bioinformatics.babraham.ac.uk/projects/trim_galore/, minimum Phred score > 25, minimum sequence length >100 nt). Exact sequence variants (ESVs) were determined from filtered sequences using DADA2 in the QIIME2 pipeline (Caporaso et al. 2010, Callahan et al. 2016). Taxonomy was assigned to ESVs by comparison to the SILVA ribosomal RNA database (Release 132). Singletons and ESVs assigned to chloroplast or mitochondrial sequences were removed from the analysis. All samples were normalized to a standard read count (n = 4273) for further analyses. 

Analyses of microbiome data were conducted separately to compare control corals and corals in direct contact or close proximity with Galaxaura or its mimics or Sargassum or its mimics. In each case, principal coordinate analysis (PCO) and corresponding tests for differences in microbiome composition (permutational multivariate analysis of variance, PERMANOVA) and variability (PERMADISP) were implemented in Primer E (Clark 1993). Alpha diversity (ESV richness, Shannon diversity) of relevant datasets was calculated using QIIME2 (Caporaso et al. 2010). Differences among treatments were analyzed using LME models in the R package nlme (Pinheiro et al. 2017) with coral colony as a random factor. When necessary, the varIdent argument was used to control for heteroscedasticity. For each analysis, significance levels were adjusted to correct for multiple comparisons of relevant datasets (? = 0.025).