2.11 Gene ontology (GO) enrichment analysis
A R package “org.Dalabrax.eg.db” specific to sea bass annotation was
generated using the MakeOrgPackage function from the AnnotationForge
Package v1.40.1. The R package ClusterProfiler version 4.6.0 (Yu et al.
2012) with the “org.Dalabrax.eg.db” was used to analyze functional
profiles of differentially methylated and expressed genes and to
identify major biological functions. The functional analysis was
performed using as input the differentially methylated genes (DMGs),
differentially methylated promoters (DMPs) and differentially expressed
genes (DEGs). A hypergeometric test was performed and enrichment p-value
of gene ontology was calculated to find significantly enriched GO terms
in the input list of DMGs, DMPs and DEGs. A p- value of
<0.05 was set as the threshold value. DMGs, DMPs and DEGs were
categorized in three categories belonging to the main GO ontologies:
biological process (BP), molecular function (MF) and cellular component
(CC). In order to study the differences in functional annotation
according to DNA methylation, we separated the DMGs and the DMPs into 4
groups: hyper-methylated gene bodies in FW- vs SW-acclimated fish
(GB hyper), hypo-methylated gene bodies (GB hypo), hyper-methylated
promoters (PR hyper) and hypo-methylated promoters (PR hypo). We
proceeded in the same way for the transcriptome by indicating genes
overexpressed in FW (up FW) or in SW (up SW). The 5 GO terms of each
analysis with the most significant p-values have been represented.
In order to highlight a functional link between changes in methylation
and expression patterns between salinity conditions, we identified the
GO terms from the methylome and transcriptome enrichment analyses.
Enriched GO terms between DEGs and DMRs (in genes and promoters) were
extracted and used to generate three Venn diagrams, one per GO term
category: BP, MF, and CC. This enabled us to identify enriched GO terms
that are common to both analyses (methylome and transcriptome).