Figure 1. Overview and performance of the MBD-SNP method.

(A) Overview of MBD-SNP workflow. Genomic DNA is fragmented by NspI and StyI in two separate reactions, and digested ends are ligated to adaptors. These products are then divided into a total input and enriched methylated fraction, the latter of which is subjected to enrichment of methylated DNA fragments by binding to MBD2-MBD immobilized magnetic beads. Both the total and enriched methylated fractions are then subjected to amplification, labeling, and hybridization to Affymetrix SNP 6.0 microarrays. Subsequent computational analyses comparing the enriched methylated and total input fractions allow assessment of total and allele-specific methylation. (B) Schematic showing generation of control specimens for testing MBD-SNP performance. (C) Receiver operator characteristic (ROC) curves for classification of total (allele-agnostic) and allele-specific methylation, generated by using CS1 (100% methylated), CS3 (0% methylated) and CS2 (50% methylated in individual specific fashion). (D) ROC curve for array performance as benchmarked against 5 loci across 44 samples verified by real-time methylation specific PCR (RT-MSP). (E) Concordance between MBD-SNP and Illumina 450k methylation estimates. There are 13,426 MBD-SNP methylation probes with an Illumina 450k probe located within 150bp. The MBD-SNP methylation score is plotted against the Illumina 450k methylation measure for each of the 13,426 probes and each of 12 specimens analyzed on both platforms. Among sites classified as unmethylated (Beta < 0.2) or highly methylated (Beta > 0.8) in the Infinium platform, 86.9% were concordant by the MBD-SNP mixture-model based classification of methylation status (p ≪ 1 × 10⁻¹⁰).