Fig. 3

Performance of MBD enrichment (MBDE) and targeted enrichment (TE) in identifying differentially methylated CpGs or regions (DMRs) in primary CRCs (vs. normal mucosa). a. Compared with TE, MBDE captured 3 times more genomic base pairs (including 33.4% of TE-captured base pairs) and almost 3 times more CpGs (including 41.3% of TE-captured sites). b. First three density plots, from left: Compared with CpGs missed by both methods (mean O:E CpG ratios: 0.35 for MBDE, 0.39 for TE), the CpGs captured by each method were preferentially located in genomic areas with relatively high CpG densities (mean O:E CpG ratios: 0.58 for MBDE, 0.65 with TE). Fourth density plot: CpGs captured by MBDE only, by TE only or by both methods tended to be located in genomic areas with similar CpG densities (mean O:E CpG ratios: 0.56 for MBDE, 0.65 for TE, 0.66 for both methods). c. Methylation changes (log fold change in MBDE, beta-values in TE) at CpGs captured by both methods in low-, medium-, and high-CpG-density areas. (See Methods for calculation of differential methylation with each method and for calculation and classification of CpG density.) Intermethod correlation values are shown for each density area. For most of the CpGs captured by both methods (gray bars), no methylation differences in primary CRCs (vs. normal mucosa) were identified with either MBDE or TE. The number of hypermethylated CpGs detected by both methods (red bars) steadily increased with increasing CpG rates. In addition, in medium- and high-CpG-rate areas of the genome, MBDE captured hypermethylated CpGs that were not found to be significantly hypermethylated with TE (blue vs. green bars). Hypomethylated CpGs were more frequently identified by TE than MBDE (yellow vs. orange bars). d. The CpG site trends were confirmed by analysis of DMRs. Over 400 hypermethylated DMRs were identified with both methods (in addition to the 1584 identified only by MBDE and the 827 detected only with TE). However, the overlap of commonly identified hypermethylated DMRs is likely underestimated because of the use of different analytical packages to identify them with MBDE or TE (see example in Supplementary Figure 3 C). Hypomethylated DMRs were in contrast identified almost exclusively by TE (examples in Supplementary Figure 3 D and E).