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!