Validation of a DNA Methylation Microarray for 850,000 CpG Sites of the Human Genome Enriched in Enhancer Sequences

Figures & data

Figure 1. Venn diagram showing the degree of CpG overlapping (yellow) between the Infinium HumanMethylation450 BeadChip (450K; red) and the MethylationEPIC BeadChip (850K; green) microarrays.

Figure 2. Genomic and functional context of the newly added 413,759 CpG sites in the MethylationEPIC BeadChip microarray (850K).

(A) Chromosome location; (B) Infinium design chemistry (Infinium I, II); (C) UCSC gene region feature category (TSS200, TSS1500, 5′UTR, 1stExon, body, 3′UTR); (D) location of the CpG relative to the CpG island (open sea, island, shore, shelf); (E) FANTOM5 associated enhancer regions; (F) ENCODE project annotation for transcription binding site; (G) open chromatin, and (H) DNase I hypersensitive regions.

Figure 3. Genomic and functional context of the 853,307 CpG sites in the MethylationEPIC BeadChip microarray (850K).

(A) Chromosome location; (B) Infinium design chemistry (Infinium I, II); (C) UCSC gene region feature category (TSS200, TSS1500, 5′UTR, 1st Exon, Body, 3′UTR); (D) location of the CpG relative to the CpG island (open sea, island, shore, shelf); (E) FANTOM5 associated enhancer regions; (F) ENCODE project annotation for transcription binding site; (G) open chromatin and (H) DNase I hypersensitive regions.

Figure 4. Technical and biological validation of the 850K MethylationEPIC microarray.

Comparison of methylation values from HumanMethylation450 and their corresponding shared CpG sites present on MethylationEPIC microarray (A) for a renal tumor sample (RCC9). Assay reproducibility (B) of methylation measurements when using technical replicates on a normal colon (NC22A) sample. Correlation plot (C) of the methylation values obtained from a FFPE sample (RCC9-FFPE) when compared with its match biopsy of the same tumor that was preserved as FF (RCC9). 5-hmC value representation (D), where the same sample was treated as per conventional bifsulfite conversion or as per oxBS. Differences on oxBS with bifsulfite conversion are due to the level of 5-hydroxymethylation, where the absence of 5-hmC has been modeled as a discontinue red line. Frequency of hydroxymethylaton values (E) as a result of subtracting the oxBS values (due to 5-mC) from the BS values (due to 5-mC + 5-hmC) for each CpG site included on the 850K array.

5-hmC: 5-hydroxymethylation; FF: Fresh frozen; FFPE: Formalin-fixed paraffin-embedded; oxBS: Oxidative bisulfite conversion.

Figure 5. Biological validation of the 850K DNA methylation microarray.

(A) Differentially methylated CpG sites (Δβ ≥0.66 ) on MethylationEPIC BeadChip microarray from a normal colon sample (NC22A), and normal sorted brain neurons (N229). Heatmap representation of differentially methylated CpG sites (left) and methylation values distribution (right) of samples where methylation differences threshold has been denoted as a discontinued red line (Δβ ≥0.66), and those values considered as differentially methylated have been highlighted in blue. (B) Differentially methylated CpG sites (Δβ ≥0.66), among the newly added 413,759 CpG sites of the MethylationEPIC BeadChip microarray, for a normal colon sample (NC22A), and normal sorted brain neurons (N229). Heatmap representation of differentially methylated CpG sites (left) and methylation values distribution (right) of samples where methylation differences threshold has been denoted as a discontinued red line (Δβ ≥0.66), and those values considered as differentially methylated have been highlighted in blue.