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Transcript of DNA Methylation Assays High Throughput Data Analysis BIOS 691-803, VCU Winter 2010 Mark Reimers,...
![Page 1: DNA Methylation Assays High Throughput Data Analysis BIOS 691-803, VCU Winter 2010 Mark Reimers, PhD.](https://reader036.fdocuments.in/reader036/viewer/2022062407/56649dd35503460f94aca2a6/html5/thumbnails/1.jpg)
DNA Methylation Assays
High Throughput Data AnalysisBIOS 691-803, VCU
Winter 2010Mark Reimers, PhD
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DNA Methylation
• Cytosine bases sometimes methylated
• Shuts down transposons
• In vertebrates:– Condenses chromatin– Renders genes inaccessible– Heritable in cell lineages– Developmental fate decisions
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DNA Methylation
Adding a Methyl to Cytosine
Cytosine methylation is passed on to daughter cells
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How Does Methylation Happen?
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Distribution of Methylation
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Distribution of CpG sites
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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
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Methylation in Development
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Methylation in Cancer
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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
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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
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MeDIP Data
EIF2C4
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Copyright restrictions may apply.
Ordway, J.M. et al. Carcinogenesis 2006 27:2409-2423; doi:10.1093/carcin/bgl161
McrBCA schematic of three array probes (X, Y and Z) arranged along a chromosome is shown
Short fragments with methylated CpG’s have been removed
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The HELP Assay
• MSPI cuts at 5’-CCGG-3’ – methylated or not
• HPAII cuts at 5’-CCGG-3’ only if unmethylated (useful restriction enzyme)
Sample
MSPI
HPAII
LabelPCR amplify
LabelPCR amplify
Co-hybridize
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HELP Data
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HELP log ratios
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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?
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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
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Normalizing Intensity Bias
• Strong intensity dependent bias in each chip
• Different intensity dependence in each chip
• Correlated with CpG density
• Ignored by BATMAN!
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Normalizing Sequence Bias
• Significant dependence of intensity on CG
• Dependence differs among chips!
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HELP ratios and fragment length
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Normalizing Fragment Length - HELP
• Distribution of intensity varies by L
• Fit density curve and line up
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More HELP technical biases
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CHARM
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Critique of CHARM• Improves reliability of mcrBC by assuming
smoothness
• Doesn’t incorporate probe effects
• No pre-processing of Illumina data used as reference
• Detailed data shows ‘block’ structure– With single CpG sites deviating from most– Difficult to detect using CHARM
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Block Structure of Methylation
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General Principles of Complex Assay Normalization
• Many reactions: each influenced by differences in processing conditions
• Differences in technique induce similar biases in probes with similar technical characteristics
• Aggregating probes by technical character (IF independent of biology) is an effective way to estimate bias on each chip individually