Fig. 7

Genome-wide single-nucleotide resolution of cytosine methylation [%] in each of the three methylated motifs (GCGC, CCTC, TCTTC) in H. pylori N6. Dot plots (A, D, F) and box plots with whiskers (median) for the three different methylated motifs (B, E, G). Panels A (GCGC), D (CCTC), and F (TCTTC) show the genome-wide single-base resolution quantitative methylation for the three cytosine motifs; the nucleotide contexts for each motif, if occurring as simple motif or overlapping combined motifs, are marked as different dot colors. Panels B, E, and G show box plots comparing the combined individual site quantitative methylation levels between single GCGC, CCTC, and TCTTC motifs, and combined quantitative information on their overlapping motifs with other, homologous or heterologous, cytosine MTase target motifs. A, B, and C include the results for the average of methylation [%] in each single-motif cytosine for all genomic GCGC motifs, stratified according to single motif, or overlapping “doublet” GCGCGC motif. Bar graph in C indicates the average methylation, combined from genome-wide doublet motif analysis, for each methylated cytosine nucleotide on both strands of the palindromic doublet GCGCGC motifs. The dashed lines in A, D, and F indicate the mean methylation level for each motif. Statistical significance for datasets shown in B, C, E, and G was calculated using the Welch two-sample t-test (**** p < 0.001). For panels A through G, conversion data from N6 wild type replicate R3 (Table 1) were analyzed (data in Additional file 3: Table S2). H Heatmap matrix plot of methylation of all GCGC motif cytosines in gene cluster crdR/crdS (encodes a Two Component System for copper sensing and resistance). For each methylated base, data from five experimental conditions in two biological replicates are depicted (see Table 1; conversion data in Additional file 3: Table S2). The heatmap illustrates the robustness of lower methylation detection by EM-Seq for the flanking motifs of overlapping doublet GCGCGC motifs in comparison with single GCGC motifs within the genomic crdS/crdR gene cluster, whose transcript activity is regulated by GCGC methylation [16]. Methylation (shown as EM-Seq conversion [%] data) of each cytosine site is shaded in different colors, coded as in the graphic legend. While the 3’-upstream single GCGC motif in the promoter region of crdR is always high-methylated (low conversion, dark blue squares), cytosines in some overlapping GCGC motifs, in particular the flanking cytosines of a completely overlapping doublet within the CDS of crdR, exhibit reproducible lower methylation (high conversion, light blue, white and reddish squares; genomic positions 1,398,590 and 1,398,593; Additional file 2: Table S1). Single-nucleotide resolution quantitative cytosine methylation in GCGC motifs in panel H was compared over different standard and cytosine methylation-influencing conditions and mutants (luxS), including at least two biological and technical replicates for each condition (Additional file 3: Table S2)