Profiling Genome-Wide DNA Methylation Patterns in Human Aortic and Mitral Valves Sarah Halawa 1,2, Najma Latif 3,4, Yuan-Tsan Tseng 3,4, Ayman M. Ibrahim 1,5, Adrian H. Chester 3,4, Ahmed Moustafa 2,6, Yasmine Aguib 1,4 and Magdi Yacoub 1,4* 1 Aswan Heart Centre, MYF, Egypt 2 Biotechnology Graduate Program, American University in Cairo, New Cairo, Egypt 3 Magdi Yacoub Institute, Heart Science Centre, Harefield, UK 4 NHLI, Imperial College London, UK 5 Zoology Department, Faculty of Science, Cairo University, 12613, Egypt 6 Department of Biology, American University in Cairo, New Cairo, Egypt * Authors to whom correspondence should be addressed 1 Contents Supplementary Figures ..................................................................................................................................................................... 4 Figure S1. Total no. of sequences for all samples before and after trimming.............................................................................. 4 Figure S2. Methylation percentage and read coverage distribution per promoter for aortic and mitral tissue samples ............ 5 Figure S3. Boxplots of % methylation per sample, clustering, heatmap and PCA based on promoter methylation for aortic and mitral tissue samples ................................................................................................................................................................... 7 Figure S4. Heatmap showing the genes associated with differentially methylated promoters and their methylation level in aortic (TA) vs. mitral tissue (TM) .................................................................................................................................................. 9 Figure S5. Additional functional categories that classify the genes associated with differentially methylated promoters in aortic vs. mitral tissue according to the PANTHER’s Pathway, Molecular Function, Biological Process, Protein Class and Cellular Component annotation sets ......................................................................................................................................... 10 Supplementary Tables .................................................................................................................................................................... 12 Table S1. Differentially methylated promoters between aortic and mitral valve tissue (q value < 0.05 and a meth.diff > |10|) and their associated genes ........................................................................................................................................................ 12 Table S2. Functional Classification based on PANTHER Pathways for the genes associated with differentially methylated promoters between the non-diseased aortic and mitral valve tissue – full list ........................................................................... 39 Table S3. Functional Classification based on PANTHER Molecular Functions for the genes associated with differentially methylated promoters between the non-diseased aortic and mitral valve tissue – full list ........................................................ 43 Table S4. Functional Classification based on PANTHER Biological Processes for the genes associated with differentially methylated promoters between the non-diseased aortic and mitral valve tissue – full list ........................................................ 46 Table S5. Functional Classification based on PANTHER Protein Classes for the genes associated with differentially methylated promoters between the non-diseased aortic and mitral valve tissue – full list ........................................................................... 49 Table S6. Functional Classification based on PANTHER Cellular Components for the genes associated with differentially methylated promoters between the non-diseased aortic and mitral valve tissue – full list ...................................................... 51 Table S7. Node characteristics of the network with the minimum interactions constructed for genes associated with differentially methylated promoters between non-diseased aortic and mitral valve tissue ....................................................... 54 Table S8. List of enriched pathways in the network constructed upon the genes associated with the differentially methylated hyo- and hypermethylated promoters between the non-diseased aortic and mitral valve tissue based on the Reactome pathway database (selected results) ........................................................................................................................................... 75 Table S9. List of enriched biological processes in the network constructed upon the genes associated with the differentially methylated hyo- and hypermethylated promoters between the non-diseased aortic and mitral valve tissue based on the Gene Ontology (GO) Biological Processes database (selected results) ................................................................................................. 80 Table S10. List of enriched molecular functions in the network constructed upon the genes associated with the differentially methylated hyo- and hypermethylated promoters between the non-diseased aortic and mitral valve tissue based on the Gene Ontology (GO) Molecular Functions database (selected results) ................................................................................................ 87 Table S11. List of enriched cellular components in the network constructed upon the genes associated with the differentially methylated hyo- and hypermethylated promoters between the non-diseased aortic and mitral valve tissue based on the Gene Ontology (GO) Cellular Components database (selected results) ............................................................................................... 92 Table S12. Node characteristics of subnetwork 1/module 1 of the NW (pvalue 3.04 e-13)........................................................ 94 Table S13. List of enriched pathways, biological processes, molecular functions and cellular components in subnetwork 1/module 1 (pvalue 3.04 e-13) of the network constructed upon the genes associated with the differentially methylated hyo- and hypermethylated promoters between the non-diseased aortic and mitral valve tissue based on KEGG/Reactome, Gene Ontology (GO) Biological Processes database, Gene Ontology (GO) Molecular Functions database and Gene Ontology (GO) Cellular Components database (full list) ..................................................................................................................................... 96 Table S14. Node characteristics of subnetwork 2/module 2 of the NW (pvalue 0.047) .............................................................. 99 2 Table S15. List of enriched pathways, biological processes, molecular functions and cellular components in subnetwork 2/module 2 (pvalue 0.047) of the network constructed upon the genes associated with the differentially methylated hyo- and hypermethylated promoters between the non-diseased aortic and mitral valve tissue based on KEGG/Reactome, Gene Ontology (GO) Biological Processes database, Gene Ontology (GO) Molecular Functions database and Gene Ontology (GO) Cellular Components database (full list) ................................................................................................................................... 100 References .................................................................................................................................................................................... 104 3 Supplementary Figures Figure S1. Total no. of sequences for all samples before and after trimming A. B. Figure S1. Total no. of sequences for A. aortic tissue (TA) samples G1T, G3T, G5T, G7T, G9T and G11T and B. mitral tissue (TM) samples G2T, G4T, G6T, G8T, G10T and G12T. “Raw” refers to the total no. of sequences processed before trimming; “Truncated” denotes sequences that were truncated to a varying degree because of deteriorating qualities (Phred score < 20); “Removed” represents sequences that were removed as they became shorter than 20 bps after trimming; “Truncated_RRBS” represents sequences trimmed by an additional 2 bp characteristic of RRBS when adapter contamination was detected; “Trimmed” refers to the total no. of sequences processed after trimming. The total number of sequences processed for samples G1T, G3T and G2T before and after trimming were significantly less compared to the rest of the samples. The threshold set to remove technical artifacts (see Methods), automatically removed these samples from our analysis ensuring that no technical bias is introduced. 4 Figure S2. Methylation percentage and read coverage distribution per promoter for aortic and mitral tissue samples 5 Figure S2. A. Methylation percentage- and B. read coverage distribution per promoter for aortic (G1T, G3T, G5T, G7T, G9T, G11T) and mitral (G2T, G4T, G6T, G8T, G10T, G12T) tissue samples. The promoter percent methylation per sample exhibited a bimodal distribution, which is expected as reads of normal cells are either methylated (100%) or unmethylated (0%) [1]. Promoter read coverage distribution did not show a secondary peak to the right, reflecting low PCR duplication levels [1]. Both distributions reflect sufficient sample quality. 6 Figure S3. Boxplots of % methylation per sample, clustering, heatmap and PCA based on promoter methylation for aortic and mitral tissue