Fig 1.
Pairwise correlation in beta values between BS and oxBS arrays.
The same sample was divided into four BS and four oxBS replicates. Beta values are generated by the minfi analysis package and represent the percentage of unconverted C’s at any given locus. The average correlation of BS replicates is 98.3 and of the oxBS replicates is 96.8. The average correlation between the two methods is 94.0.
Fig 2.
Effect of number of replicates on 5hmC detection.
Top left: Number of probes detected as differentially methylated, i.e. as hydroxymethylated (having a positive 5hmC signal), with all four replicates and with only replicates 1 and 3. Top right: Using only replicates 2 and 4. The number of probes observed in two replicates but not in four fall within the expected 1% FDR. Bottom: Probes with 5hmC in pair 1 and 3 compared to pair 2 and 4. Only 38,246 probes were observed in both pairs.
Fig 3.
Only probes which were significantly differentially methylated between BS and oxBS are shown hence the lack of probes around zero. For the plot of all probes see S5 Fig. Negative values fall within the expected 1% FDR.
Fig 4.
Top: the number of probes in each genomic region in the entire 450K array.
The highest number of probes are in introns, followed by intergenic regions. Bottom: the proportion of probes in each region found to have a positive 5hmC signal. While only a small number of probes are designed in 3’UTR’s (12984), this region has the highest proportion of probes with a positive 5hmC signal (47%). Regions labeled “Other” are those that do not fit into any of the previous categories.
Fig 5.
Distribution of 5mC (blue) and 5hmC (pink) levels across regions.
The lowest median levels of both are in 5’UTR’s, however, beyond that there is little correlation between 5mC and 5hmC levels. Median 5hmC levels are similar across all regions, while 5mC levels show greater variation.
Fig 6.
Genomic distribution of 5mC and 5hmC.
6a: Distribution of 5hmC and 5mC levels at CpG islands, shores (0–2 kb) and shelves (2–4 kb). 5mC levels show a steep rise moving away from CpG sites, 5hmC levels are lowest at CpG sites but show less variation between shores and shelves. The width of the boxes is proportional to the number of underlying probes. 6b: Distribution of 5mC and 5hmC levels around the TSS. Levels of both types of methylation rise steeply moving away from TSS’s.
Fig 7.
5hmC levels are highest at intermediate levels of 5mC (~40%).
Fig 8.
Comparison between oxBS arrays and qPCR.
Right: Correlation between 5hmC detected by qPCR vs array probes. Left: Discrepancy between qPCR and arrays quantified as percentage difference relative to qPCR (See also S1 Document). Data points having the same coordinates appear in darker red.