2.4 Microbiological analysis
Genomic DNA was extracted from a 0.5 g frozen coral sample using the
TGuide S96 magnetic bead method and the DP812 DNA extraction kit
(Tiangen Biotech Co., Ltd., Beijing, China). The V3-V4 variable region
of the bacterial 16S rRNA gene was PCR amplified with the forward primer
27 F (5’-AGRGTTTGATYNTGGCTCAG-3’) and the reverse primer 1492 R
(5’-TASGGHTACCTTGTTASGACTT-3’) (Zhong et al., 2021). Library preparation
was performed using the SMRTbell Template Prep Kit (PacBio, USA), and
the PCR reaction was conducted in a Veriti 96-well thermal cycler
(Applied Biosystems, USA). Sequencing was carried out on the Sequel II
platform after purification.
After obtaining the sequencing data, the raw reads underwent filtering
to remove adapter sequences using Trimmomatic (version 0.33) and
Cutadapt (version 1.9.1) software (Bolger et al., 2014; Martin, 2011) to
obtain clean reads. Paired reads were merged using USEARCH (version 10)
(Edgar, 2013), and chimeric sequences were removed using UCHIME (version
8.1). Subsequently, high-quality sequences were obtained for further
analysis. The sequences were clustered at a 97 % similarity level using
USEARCH (version 10.0) with a default OTU filtering threshold of 0.005
% of the total sequencing count. For ASV analysis, the DADA2 method in
QIIME2 (version 2020.6) was employed to denoise the data, with the
default filtering threshold of 0.005 % of the total sequencing count.
The obtained high-quality sequences were then annotated against
databases such as Silva, Unite, Greengenes, NCBI, Fungene, and MaarjAM
for taxonomic classification and functional prediction using BugBase and
FAPROTAX. Alpha and beta diversity analyses, community composition
analyses, and BugBase and FAPROTAX functional prediction analyses were
conducted using BMKCloud (www.biocloud.net). Core OTU analysis was
carried out using Venn diagrams to identify shared OTUs among different
sample groups, and the composition and relative abundance differences of
core OTUs between groups were analyzed using pie charts and heatmaps.
Data visualization and figure generation were performed using GraphPad
Prism 8.0.2 and RStudio. The data that support the findings of this
study are openly available in figshare:
https://doi.org/10.6084/m9.figshare.24152901.