3.1 DNA methylation patterns in contrasted salinity treatments
Sequencing of the samples yielded 1 205 785 497 bp Illumina paired-end
reads, with an average of 120 578 550 paired-end reads by library. After
quality control and alignment, an average of 90 866 384 (75.4%) reads
per library was uniquely mapped (Table 1S) and only the uniquely aligned
reads were submitted for subsequent analyses. For each sample, the
bisulfite conversion efficiency was larger than 98.7 % (Table 1S). For
all samples, the majority of methylated cytosines were in the CpG
context. The average methylation of CpG sites was 74.38 % ± 1.97
% in the SW control group and 73.84 % ± 2.31 % in FW-exposed D.
labrax (Fig. 1A). Even if there was no significant difference in CpG
methylation levels between FW and SW samples (Mann Whitney test,p = 0.83) (Fig. 1A), there seemed to be a trend of FW samples
being less methylated than SW samples, which was confirmed in subsequent
analyses (see subsection ‘The effect of freshwater transfer on DNA
methylation dynamics’). DNA methylation levels were the lowest in the
promoter region at the transcription start site (TSS) and the 5’UTR
(untranslated region). Higher DNA methylation levels were observed in
the exons and introns. Downstream the transcription end sites (TES), a
slightly lower DNA methylation was measured than in gene bodies (Fig.
1A, B).
A hierarchical clustering (Fig. 2A) and a principal component analysis
(PCA) (Fig. 2B) were performed from methylation level per base data, in
order to identify patterns in genome-wide methylation across treatment
groups and between individuals from the same condition. Fish exposed to
fresh water (black circles, Fig. 2A) clustered distinctly from fish
exposed to seawater (white circles) except two individuals (SW-2 and
FW-4). Individual PCA based on DNA methylation patterns is shown in Fig.
2B. Principal component 1 (PC1) explained 13.6 % of the variance of the
data whereas PC2 explained 12.3 %, which makes a cumulative percentage
of 25.9 %. Based on their CpG methylation profiles, distinct patterns
between FW and SW exposed fish were observed with FW-acclimated fish
appearing at the left upper part of the component pattern plot and
SW-acclimated fish at the right lower part of the component pattern
plot.