Metagenomic analysis
Microbial DNA from the coral mucus collected on the 0.22-µm Sterivex was
extracted using a modified Macherey-Nagel protocol using NucleoSpin
column for purification. DNA was stored at - 20°C until quantification
with Qubit (Thermo Fisher Scientific) (Dinsdale et al. 2008b). The Swift
kit 2S plus (Swift Biosciences) was used for library preparation since
it provides good results from small amounts of input DNA, characteristic
of microbial samples collected from the surface of the host (Doane et
al. 2017; Cavalcanti et al. 2018). All samples were sequenced by the
Dinsdale lab on Illumina MiSeq at San Diego State University. The
sequenced DNA was analyzed for quality control using PrinSeq (Schmieder
and Edwards 2011) before annotation. The metagenomes were annotated
through MG-RAST (Meyer et al. 2019), using the RefSeq database for
taxonomic annotations and the SEED database for functional annotations.
For the taxonomic composition, the metagenomes were filtered in MG-RAST
to include only Bacteria at the level of genera. For the functional
composition, the metagenomes were filtered in MG-RAST for Stress
Response, Nitrogen Metabolism, and Sulfur Metabolism. We selected these
three broad functional gene groups (SEED subsystem level 1) because they
have the greatest relevance for corals under heat stress (Wegley et al.
2007; Thurber et al. 2009; Littman et al. 2011; Raina et al. 2013;
Nguyen-Kim et al. 2015; Cardini et al. 2016; Pogoreutz et al. 2017; Wang
et al. 2018; Sun et al. 2022). The number of sequence hits for each
microbial taxon or function is represented as the relative abundance by
calculating the proportion of sequence hits for that parameter over the
total number of sequences annotated for that metagenome. Metagenomes
were compared using proportional abundance, which is preferred to
rarefaction (McMurdie and Holmes 2014; Quince et al. 2017; Luz Calle
2019). Bacteria accounted for approximately 99 % of the annotation;
therefore, we are only analyzing bacterial taxa and gene functions in
this study. Metagenomes were sequenced from a subset of the coral
nubbins used in the experiment (n = 8), representing four different
coral colonies (n = 2 colonies per reef zone) that had their mucus
sampled before and after the experimental treatments (n = 16 metagenomes
total). These metagenomes were compared with the ones from the same
colonies collected in situ (Lima et al. 2022).