DNA Methylation Assays Virginia Commonwealth DNA甲基化检测弗吉尼亚联邦.pptVIP

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DNA Methylation Assays Virginia Commonwealth DNA甲基化检测弗吉尼亚联邦.ppt

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DNA Methylation Assays High Throughput Data Analysis BIOS 691-803, VCU Winter 2010 Mark Reimers, PhD DNA Methylation Cytosine bases sometimes methylated Shuts down transposons In vertebrates: Condenses chromatin Renders genes inaccessible Heritable in cell lineages Developmental fate decisions DNA Methylation How Does Methylation Happen? Distribution of Methylation Distribution of CpG sites DNA Methylation and Transcription Methyl groups block access to some transcription factors Me-C attracts MBD proteins that further suppress transcription Heavy methylation predisposes chromatin to condense Methylation in Development Methylation in Cancer Assaying Methylation MeDIP (Methylated DNA immuno-precipitation) Antibody to Me-C = ChIP – chip Doesn’t distinguish among nearby sites Multiple restriction enzyme assays Isoschizomer (HpaII/MspI) assays: MIAMI (Microarray-based Integrated Analysis of Methylation by Isoschizomers) HELP Bisulphite conversion of meC - T, then hybridize to SNP style array MeDIP Genomic DNA is randomly sheared by sonication Immunoprecipitate with an antibody that specifically recognizes 5-methylcytidine (5mC) Hybridize against control (no antibody) on array MeDIP Data The HELP Assay MSPI cuts at 5’-CCGG-3’ methylated or not HPAII cuts at 5’-CCGG-3’ only if unmethylated (useful restriction enzyme) HELP Data HELP log ratios Methylation Data Analysis Regional QA Normalizing Bias in ratios Probe sequence CpG density Intensity Fragment length (for HELP similar) Estimation Are methylations similar at neighbors? Normalizing MeDIP – CpG Bias Direct approach Compare to a standard: fully methylated Tanay: M.SssI treatment Indirect estimate Regress M (ratio) on CpG density (assuming all neighbors are equal) Down et al: BATMAN Normalizing Intensity Bias Strong intensity dependent bias in each chip Different intensity dependence in each chip Correlated with CpG density Ignored by BATMAN! Normalizing Sequence Bias Significant dependence of intensity on