Fig. 3: The identification and validation of BC-associated DNA methylation markers.

a The box plot shows the β-value distribution of eight candidate methylation markers in PBMCs from BC patients (n = 50) and normal controls (n = 30). A β-value of zero represents no methylation, and 1 represents full methylation. P values were determined by two-sided Mann–Whitney U-test. ***p ≤ 0.001; ****p ≤ 0.0001. b The box plots present the β-value distribution of four methylation markers validated by pyrosequencing (top, 50 BC and 50 normal controls) and targeted bisulfite sequencing (bottom, 60 BC and 40 normal controls) in PBMCs samples. P values were determined by a two-sided Mann–Whitney U-test. ***p ≤ 0.001; ****p ≤ 0.0001. c The methylation levels of four methylation markers in 40 normal controls and 60 BC patients of the targeted bisulfite sequencing set with different age, stage, tumor size, lymph node metastasis status, ER status, PR status, HER2 status, and Ki67 levels (one-Way ANOVA test, two-sided, Dunnett’s test for multiple comparisons). *p ≤ 0.05; ***p ≤ 0.001; NS no significance. d ROC curves of four methylation markers in all BC, stage 0/I BC and ≤1.5 cm BC of the targeted bisulfite sequencing set. The AUCs for the different categories are shown in the legend. e Unsupervised hierarchical clustering of four methylation markers based on differential methylation levels between BC patients and normal controls in the targeted bisulfite sequencing set. The boxes in a–c are bounded by the first and third quartile with a horizontal line at the median; minima is the smallest data greater than or equal to the first quartile – 1.5 × interquartile range (IQR); maxima is the largest data point less than or equal quartile + 1.5 × IQR. Source data are provided as a Source Data file.