Download - 4 - DNA Methylation Assays
-
7/28/2019 4 - DNA Methylation Assays
1/27
DNA Methylation Assays
High Throughput Data Analysis
BIOS 691-803, VCU
Winter 2010Mark Reimers, PhD
-
7/28/2019 4 - DNA Methylation Assays
2/27
DNA Methylation
Cytosine bases sometimes methylated
Shuts down transposons
In vertebrates: Condenses chromatin
Renders genes inaccessible
Heritable in cell lineages Developmental fate decisions
-
7/28/2019 4 - DNA Methylation Assays
3/27
DNA Methylation
Adding a Methyl to Cytosine
Cytosine methylation is passed on to daughter cells
-
7/28/2019 4 - DNA Methylation Assays
4/27
How Does Methylation Happen?
-
7/28/2019 4 - DNA Methylation Assays
5/27
Distribution of Methylation
-
7/28/2019 4 - DNA Methylation Assays
6/27
Distribution of CpG sites
-
7/28/2019 4 - DNA Methylation Assays
7/27
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
-
7/28/2019 4 - DNA Methylation Assays
8/27
Methylation in Development
-
7/28/2019 4 - DNA Methylation Assays
9/27
Methylation in Cancer
-
7/28/2019 4 - DNA Methylation Assays
10/27
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
-
7/28/2019 4 - DNA Methylation Assays
11/27
MeDIP
Genomic DNA israndomly sheared bysonication
Immunoprecipitatewith an antibody thatspecificallyrecognizes 5-methylcytidine (5mC)
Hybridize againstcontrol (no antibody)on array
-
7/28/2019 4 - DNA Methylation Assays
12/27
MeDIP Data
EIF2C4
-
7/28/2019 4 - DNA Methylation Assays
13/27
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
-
7/28/2019 4 - DNA Methylation Assays
14/27
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
-
7/28/2019 4 - DNA Methylation Assays
15/27
HELP Data
-
7/28/2019 4 - DNA Methylation Assays
16/27
HELP log ratios
-
7/28/2019 4 - DNA Methylation Assays
17/27
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?
-
7/28/2019 4 - DNA Methylation Assays
18/27
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
-
7/28/2019 4 - DNA Methylation Assays
19/27
Normalizing Intensity Bias
Strong intensity
dependent bias in
each chip
Different intensitydependence in each
chip
Correlated with CpGdensity
Ignored by BATMAN!
-
7/28/2019 4 - DNA Methylation Assays
20/27
Normalizing Sequence Bias
Significant dependence of intensity on CG
Dependence differs among chips!
-
7/28/2019 4 - DNA Methylation Assays
21/27
HELP ratios and fragment length
-
7/28/2019 4 - DNA Methylation Assays
22/27
Normalizing Fragment Length -
HELP Distribution of intensity varies by L
Fit density curve and line up
-
7/28/2019 4 - DNA Methylation Assays
23/27
More HELP technical biases
-
7/28/2019 4 - DNA Methylation Assays
24/27
CHARM
C i i f CHARM
-
7/28/2019 4 - DNA Methylation Assays
25/27
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
-
7/28/2019 4 - DNA Methylation Assays
26/27
Block Structure of Methylation
-
7/28/2019 4 - DNA Methylation Assays
27/27
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