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.