Résumé
Methylome of the fresh water snail Biomphalaria glabrata. DNA was extracted from the feet of 10 individuals of B. glabrata originally isolated from Brazil. These snails have been cultivated in the laboratory since 1960. Tissue were grinded at 4°C and incubated in 1 ml volume of lysis buffer (20 mM TRIS pH 8; 1 mM EDTA; 100 mM NaCl; 0.5% SDS), with 0.3 mg of proteinase K at 55°C for 1 night. Afterwards, lysate was purified with phenol-chloroform and DNA was isopropanol precipitated. The extracted DNA (around 138ng/µL) was poled in equivalent amounts and Whole Genome Bisulfite Sequencing was done by GATC-biotech (www.gatc-biotech.com). The principle of this treatment is to convert non-methylated cytosines of gDNA into deoxy-uracil, whereas methylated cytosines remain intact. WGBS was done according to the Lister protocol (sequence 2 forward strands only). The reference genome (Biomphalaria-glabrata-BB02_SCAFFOLDS_BglaB1.fa) and annotation (Biomphalaria-glabrata-BB02_BASEFEATURES_BglaB1.3.gff3) used in this project are available on VectorBase (https://www.vectorbase.org/). To align our short reads, we chose to use two specific bisulfite mapping tools, BSMAP 1.0.0 (https://code.google.com/p/bsmap/) and Bismark 0.10.2 (www.bioinformatics.babraham.ac.uk /projects/bismark/), to compare their efficiency and convenience to finally work with the more suitable one on our datasets. IGV (Interactive Genomics Viewer, https://www.broadinstitute.org/igv/) was used to visualized final alignments. BSMAP performed better than Bismark and was used for downstream analyses. Without default parameters alignement efficiency for BSMAP is 47.1%, allowing for 2 mismatches increases it to 55.6%. Methylation occurs predominantly in CpGs. (C methylated in CpG context: 12.4%, C methylated in CHG context: 0.5%, C methylated in CHH context: 0.5%) The major part of CpG sites, 95.7% were unmethylated, of the remaining 4.3% of CpG sites around 3.8% had low methylation, and 0.5% were completely methylated. Methylation is of the mosaic type. Methylation is relatively low with 1.2% of total cytosines. Our analyses suggested that conserved genes and genes with stable expression are localized in high methylated regions of the genome. Finally, we see that repetitive sequences were predominantly situated in low methylated regions of B. glabrata. Wiggle files were generated for CpG pairs only. Produced at IHPE (http://ihpe.univ-perp.fr/)