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Principles and challenges of DNA methylation profiling
Department of Forensic Medicine Yonsei University College of Medicine
정 상 은
Epigenetics
DNA methylation
DNA methylation is an epigenetic mechanism used by cells to control gene expression.
A suitable method for DNA methylation analysis
Human Methylation450 BeadChip
Pyrosequencing
MSP (Methylation Specific PCR/qPCR)
Capillary Electrophoresis (CE) System
NGS
DNA Methylation Analysis Method
DNA Methylation Analysis (Bisulfite conversion)
Human Methylation 450 BeadChip
- 8 -
Human Methylation 450 Provides coverage.
Human Methylation 450 Workflow
Human Methylation 450 Data analysis
Background Normalization Filtering Out CpG site with
detection p.value >0.05
Correlation Quantile Normalization Data Transformation
: Avg_beta value, M value, log2(Avg_beta) value
Analysis
Image analysis
Data pre-processing
Criteria (연구자 변경 가능) 1) Spearman Correlation Test p.value < 0.05 2) |delta_mean| > 0.3 3) T-test p.value < 0.05 4) OddsRatio < 0.5 or OddsRatio > 2 5) |Fold Change| ≥ 2
Pyrosequencing
MSP (Methylation Specific PCR/qPCR)
Capillary Electrophoresis (CE) System
NGS : Whole-Genome Bisulfite Sequencing
Human Methylation450 BeadChip
DNA Methylation analysis Workflow
Significant CpG sites
Choosing a suitable method
Primer Design
Detection
Data analysis
Human Methylation450 BeadChip Pyrosequencing MSP (Methylation Specific PCR/qPCR) Capillary Electrophoresis (CE) System NGS Cloning and sequencing
Human Methylation450
BeadChip
Significant CpG sites
Choosing a suitable method
Primer Design
Detection
Data analysis
Human Methylation450
BeadChip
Significant CpG sites
Choosing a suitable method
Primer Design
Detection
Data analysis
< Body Fluid Identification>
Human Methylation450
BeadChip
Significant CpG sites
Choosing a suitable method
Primer Design
Detection
Data analysis
Selection criteria No.probe
Quality-filtered probes 485,386
p < 0.05 74,807
p < 0.05 & R-squared > 0.65 79
p < 0.05 & R-squared >0.65 & diff >=0.1 61
Age-prediction model
Human Methylation450 BeadChip
Significant CpG sites
Choosing a suitable method
Primer Design
Detection
Data analysis
Human Methylation450 BeadChip Pyrosequencing MSP (Methylation Specific PCR/qPCR) Capillary Electrophoresis (CE) System - Single base extension (SBE) NGS Cloning and sequencing
Human Methylation450
BeadChip
Significant CpG sites
Choosing a suitable method
Primer Design
Detection
Data analysis
http://genome.ucsc.edu/index.html
Human Methylation450
BeadChip
Significant CpG sites
Choosing a suitable method
Primer Design
Detection
Data analysis
http://genome.ucsc.edu/cgi-bin/hgBlat
Human Methylation450
BeadChip
Significant CpG sites
Choosing a suitable method
Primer Design
Detection
Data analysis
Human Methylation450
BeadChip
Significant CpG sites
Choosing a suitable method
Primer Design
Detection
Data analysis
Human Methylation450
BeadChip
Significant CpG sites
Choosing a suitable method
Primer Design
Detection
Data analysis
http://www.urogene.org/cgi-bin/methprimer/methprimer.cgi
Bisulfite sequencing
Human Methylation450
BeadChip
Significant CpG sites
Choosing a suitable method
Primer Design
Detection
Data analysis
Human Methylation450
BeadChip
Significant CpG sites
Choosing a suitable method
Primer Design
Detection
Data analysis
http://www.urogene.org/cgi-bin/methprimer/methprimer.cgi
MSP
Human Methylation450
BeadChip
Significant CpG sites
Choosing a suitable method
Primer Design
Detection
Data analysis
Human Methylation450
BeadChip
Significant CpG sites
Choosing a suitable method
Primer Design
Detection
Data analysis
Bisulfite converted sequence (CG YG, C T)
Human Methylation450
BeadChip
Significant CpG sites
Choosing a suitable method
Primer Design
Detection
Data analysis
http://probes.pw.usda.gov/cgi-bin/batchprimer3/batchprimer3.cgi
Human Methylation450
BeadChip
Significant CpG sites
Choosing a suitable method
Primer Design
Detection
Data analysis
Human Methylation450
BeadChip
Significant CpG sites
Choosing a suitable method
Primer Design
Detection
Data analysis
Methylation SNaPshot System
GA
Me Un
CT
Me
Un FORWARD REVERSE
Human Methylation450
BeadChip
Significant CpG sites
Choosing a suitable method
Primer Design
Detection
Data analysis
IBM SPSS Statistics 20
Human Methylation450
BeadChip
Significant CpG sites
Choosing a suitable method
Primer Design
Detection
Data analysis
IBM SPSS Statistics 20
Thank you for your attention!
