4 - DNA Methylation Assays

7/28/2019 4 - DNA Methylation Assays

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DNA Methylation Assays

High Throughput Data Analysis

BIOS 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 MBDproteins 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

Doesnt distinguish among nearby sites

Multiple restriction enzyme assays Isoschizomer (HpaII/MspI) assays:

MIAMI (Microarray-based Integrated Analysis ofMethylation by Isoschizomers)

HELP Bisulphite conversion of meC -> T, then

hybridize to SNP style array


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MeDIP

Genomic DNA israndomly sheared bysonication

Immunoprecipitatewith an antibody thatspecificallyrecognizes 5-methylcytidine (5mC)

Hybridize againstcontrol (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 CpGs

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 intensitydependence in each

chip

Correlated with CpGdensity

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

C i i f CHARM


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Critique of CHARM

Improves reliability of mcrBC by assuming

smoothness

Doesnt 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 similarbiases 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

Documents

4 - DNA Methylation Assays