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When comparing the overall amount of cytosine methylation from the three time points, the majority of reads showed no significant change over the course of the experiment, which is in agreement with data generated using the Illumina platform (Supplementary Figure S1). However, the percentage of reads with significant methylation changes in the early stages of the time course was higher in bisulfite sequencing compared to MeDIP sequencing. Also, in bisulfite sequencing, a large number of cytosines were identified (on average 6%), which could not be detected by MeDIP and vice versa (Supplementary Figure S4). However, the majority of such sites were found in the 35S promoter region and thus were excluded from the subsequent analysis. The percentage of remethylated cytosines was also higher for the bisulfite-sequencing coverage compared to the MeDIP-sequencing coverage (Supplementary Figure S4).
Our model allows correction of methylation changes towards the methylated and unmethylated states. Overall, the genome-wide cytosine methylation level increased at each time point as shown by both, the average methylation level and the variance (Figure 1). However, neither the average methylation level nor the variance of the samples changed significantly over time. In contrast, methylation decreased at CpG sites located in the 35S promoter region while the average methylation level and variance were both significantly lower in the samples at day 2 and 3 than in the samples at day 0 (Figure 1). Also, the cytosines in the 35S promoter region were remethylated more efficiently than methylated cytosines, resulting in a hypermethylation of the 35S promoter region at the later time points of the time course (Figure 1). Interestingly, in DMC P-value plots, the hypermethylated CpG sites in the 35S promoter region were more sensitive to conversion than methylated (Supplementary Figure S5), whereas the difference was less evident for MeDIP-sequencing data (Supplementary Figure S6). d2c66b5586
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