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Additional file 1: Fig. S1. Flowchart describing data and the different steps of the analysis leading to the identification of 89,118 miRNA-methylation Quantitative Trait Loci (mimQTLs). Examples of negative and positive correlation between methylation at a CpG and expression of a miRNA are shown as scatterplots at the bottom. Fig. S2. Overview of the 89,118 miRNA-CpG associations found significant in both cohorts. The scatterplots show a) the –log10(Bonferroni-adjusted Spearman correlation p-values) of Oslo2 (x-axis) vs. TCGA (y-axis); b) Spearman correlation coefficients in Oslo2 (x-axis) vs. TCGA (y-axis). The histograms show the distribution of the correlation coefficients (Spearman’s rho) of all significant miRNA-CpG correlations in the Oslo2 (c) and TCGA (d) cohorts. Fig. S3. Number of associations and genomic positions of mimQTL miRNAs and CpGs. a) Barplot showing the number of negative (neg) and positive (pos) CpG correlations (cor) for the three different miRNA clusters. b) mimQTL Manhattan plot with genomic coordinates of CpGs (black or gray) and miRNAs (green) displayed along the x-axis, with the negative logarithm of the Bonferroni-corrected Spearman correlation p-value from Oslo2 on the y-axis. Each dot on the plot signifies a CpG or miRNA (CpGs are shown in two colors to distinguish the chromosomes more clearly). c) In cis mimQTL Manhattan plot displaying the chromosomal location (using the position of the CpG) along the x-axis of the 5125 mimQTLs found on the same chromosome (in cis). Each dot represents one mimQTL which is color-coded according to negative (black) or positive (green) miRNA-CpG correlation. The y-axis displays the negative logarithm of the Bonferroni-corrected Spearman correlation p-value from Oslo2. Fig. S4. Barplots showing the number of associations per miRNA or CpG. a) Barplot showing the number of CpG associations per miRNA (n = 119). Note that the y-axis is on log scale. b) Barplot showing the number of miRNA associations per CpG (n = 26,746). Fig. S5. Density plots showing the degree of CpG co-methylation or miRNA co-expression between cluster members (see Fig. 1 and Additional file 3 a, b) calculated by Spearman correlation. a) Correlation between CpG cluster members in the Oslo2 data. b) Correlation between CpG cluster members in the TCGA data. c) Correlation between miRNA cluster members in the Oslo2 data. d) Correlation between miRNA cluster members in the TCGA data. The dotted lines represent density plots of corresponding correlations expected by chance, i.e. correlations observed after randomly permuting the same data before performing correlation analyses. Fig. S6. Density plots showing the distribution of Spearman correlation coefficients between miRNA expression and selected variables for members of each of the miRNA clusters. a, b) miRNA expression-immune infiltration score [34] correlations for the Oslo2 (a) and TCGA (b) cohorts. c, d) miRNA expression-fibroblast infiltration score [35] correlations for the Oslo2 (c) and TCGA (d) cohorts. e, f) miRNA expression-ESR1 mRNA expression correlations for the Oslo2 (e) and TCGA (f) cohorts. Fig. S7. Heatmaps showing hierarchical clustering of miRNA expression levels (rows) from tumors (columns) of the Oslo2 (top) and TCGA (bottom) cohort. Clustering was performed using Euclidean distance and average linkage. Tumors are annotated with the following clinical/molecular classifications: PAM50 molecular subtypes (Luminal A (LumA), Luminal B (LumB), Basal-like (Basal), HER2-enriched (Her2), Normal-like (Normal); Lymphocyte infiltration (LI) group where tumors were divided into quartiles: 1 (low) – 4 (high); Fibroblast infiltration group (Fibro) where tumors were divided into quartiles: 1 (low) – 4 (high); Human epidermal growth factor receptor 2 (HER2) status; Estrogen receptor (ER) status. a, b) Clustering of miRNA cluster A expression (n = 23); c, d) Clustering of miRNA cluster B expression (n = 59); e, f) Clustering of miRNA cluster C expression (n = 37). Fig. S8. Heatmaps showing hierarchical clustering of methylation levels of CpG cluster 1 (a; n = 14,040) and CpG cluster 2 (b; n = 12,706) in the TCGA cohort (CpGs in rows and tumors in columns). Clustering was performed using Euclidean distance and average linkage. Tumors are annotated with the following clinical/molecular classifications: PAM50 molecular subtypes (Luminal A (LumA), Luminal B (LumB), Basal-like (Basal), HER2-enriched (Her2), Normal-like (Normal); Lymphocyte infiltration (LI) group where tumors were divided into quartiles: 1 (low) – 4 (high); Human epidermal growth factor receptor 2 (HER2) status; Estrogen receptor (ER) status. The CpGs are annotated according to overlap with regions annotated as “active intergenic enhancer” from ChromHMM of subtype-specific cell lines [37] with corresponding subtype colors. Fig. S9. Boxplot showing average DNA methylation of CpGs from cluster 1 in PAM50 subtypes of the Oslo2 cohort (Luminal A (LumA), Luminal B (LumB), Basal-like (Basal), HER2-enriched (Her2)). b) Boxplot showing average DNA methylation of CpGs from cluster 2 in the TCGA cohort when tumors were separated into quartile lymphocyte infiltration groups from low (1) to high (4) infiltration. c) Boxplot showing average DNA methylation of CpGs from cluster 2 in normal breast tissue (reduction mammoplasty, n = 17) or estrogen receptor (ER) positive (pos) or negative (neg) tumors of the Oslo2 cohort. d) Boxplot showing average DNA methylation of CpGs from cluster 2 in normal breast tissue (normal adjacent breast tissue, n = 97) or ER positive or negative tumors of the TCGA cohort. P-values resulting from Kruskal-Wallis tests indicated. Fig. S10. Boxplot showing DNA methylation of the hub CpG of miRNA cluster A (cg14270581; y-axis)) in ER positive (pos) and negative (neg) breast cancer cell lines and from different immune cell types (x-axis); B-cells, leukocytes (leuko), monocytes (mono) and T-cells. P-value resulting from Wilcoxon rank-sum test between cancer cell lines vs. immune cells is indicated. Fig. S11. Density plot showing the distribution of the Global Methylation Alteration (GMA) score in normal adjacent breast tissue (green), tumors (black) and tumors separated into estrogen receptor (ER) positive (pos) and negative (neg). Data from TCGA. Fig. S12. Top panel: Scatterplots showing on the x-axis mRNA expression of DNMT3A (left), DNMT3B (middle) and DNMT1 (right) vs. hsa-miR-29c-5p expression (y-axis) measured in 377 samples of the Oslo2 cohort. Bottom panel: Scatterplots showing on the x-axis protein expression of DNMT3A (left), DNMT3B (middle) and DNMT1 (right) vs. hsa-miR-29c-5p expression (y-axis) measured in 45 samples of the Oslo2 cohort. Each dot represents a tumor color-coded according to PAM50 subtype (Luminal A (LumA): dark blue; Luminal B (LumB): light blue; Basal-like (Basal): red; HER2-enriched (Her2): pink; Normal-like: green). Spearman correlation coefficient and p-value are indicated for each plot.