2.9 Differential methylation analysis
Differential methylation analyses were performed using the DMRseq
package (version 1.18.0, Korthauer 2017) with the following parameters:
block = FALSE, min Num Region = 5, deltamax = 0.25, bp span = 1000, min
In Span = 10, max Gap Smooth = 2500, smooth = TRUE). DMRseq output
regions (grouped per 1000 bp) were considered as differentially
methylated regions (DMRs) when the p-value was ≤ 0.05. Distribution of
DMRs across genome features (promoter, gene body, exons, introns and
intergenic regions) was performed using the bedtools intersect function
(Quinlan, 2014) with the position of each genomic feature from the sea
bass genome annotation file (Tine et al., 2014). DMRs located between
two genomic features were counted as being located in both regions. The
promoter position was defined 2kb upstream the Transcription Start Site
(TSS). Hyper- and hypomethylated regions were determined according to
the beta value from the DMRseq output file where a positive and a
negative value correspond respectively to hyper- and hypomethylation in
fresh water vs seawater. To visualize the chromosomal
distribution of the DMRs, an horizontal bar plot was generated, showing
the percentage of hyper- and hypomethylated regions on all D.
labrax chromosomes. To determine the distribution of the DMRs within
the genes among different exons and introns, we compared DMRs in exons 1
to 10 and last exons of genes as well as introns 1-10 and last introns.
The analysis was performed on 10 exons as this is the average number of
exons in D. labrax genes. Genes with only one exon were not
included in the analysis. The sum of hypermethylated regions in all
first exons was normalized by the total length (in bp) of all first
exons, then this ratio was multiplied by 100 to get a percentage. The
same method was used to determine a percentage of hypo- and
hypermethylated regions in the other exons and introns 1-10, as well as
last introns. This method enables to compare DMR frequency and
distribution between different exons and introns and prevents bias due
to the difference in length among exons and introns.