Principles and challenges of DNA methylation profiling Department of - PowerPoint PPT Presentation

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

DNA Methylation Analysis Method  Human Methylation450 BeadChip  Pyrosequencing  MSP (Methylation Specific PCR/qPCR)  Capillary Electrophoresis (CE) System  NGS

DNA Methylation Analysis (Bisulfite conversion)

Human Methylation 450 BeadChip

Human Methylation 450 Provides coverage. - 8 -

Human Methylation 450 Workflow

Human Methylation 450 Data analysis Background Normalization Image analysis Filtering Out CpG site with detection p.value >0.05 Correlation Data pre-processing Quantile Normalization Data Transformation : Avg_beta value, M value, log2(Avg_beta) value Criteria ( 연구자 변경 가능 ) 1) Spearman Correlation Test p.value < 0.05 2) |delta_mean| > 0.3 Analysis 3) T-test p.value < 0.05 4) OddsRatio < 0.5 or OddsRatio > 2 |Fold Change| ≥ 2 5)

Pyrosequencing

MSP (Methylation Specific PCR/qPCR)

Capillary Electrophoresis (CE) System

NGS : Whole-Genome Bisulfite Sequencing

DNA Methylation analysis Workflow Human Methylation450 BeadChip Significant CpG sites Choosing a suitable method  Human Methylation450 BeadChip  Pyrosequencing  MSP (Methylation Specific PCR/qPCR) Primer Design  Capillary Electrophoresis (CE) System  NGS  Cloning and sequencing 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

Age-prediction model Human Methylation450 BeadChip Selection criteria No.probe Quality-filtered probes 485,386 p < 0.05 74,807 Significant CpG sites p < 0.05 & R-squared > 0.65 79 p < 0.05 & R-squared >0.65 & diff >=0.1 61 Choosing a suitable method Primer Design Detection Data analysis

Human Methylation450 BeadChip Significant CpG sites Choosing a suitable method  Human Methylation450 BeadChip  Pyrosequencing  MSP (Methylation Specific PCR/qPCR) Primer Design  Capillary Electrophoresis (CE) System - Single base extension (SBE)  NGS Detection  Cloning and sequencing 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 Human Methylation450 BeadChip Significant CpG sites Bisulfite Choosing a sequencing 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 Human Methylation450 BeadChip Significant CpG sites Choosing a suitable method MSP 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

Human http://probes.pw.usda.gov/cgi-bin/batchprimer3/batchprimer3.cgi 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 Methylation SNaPshot System Methylation450 BeadChip Significant CpG sites Choosing a suitable method Primer Design Detection REVERSE FORWARD Un Un Me Me CT GA Data analysis

Human IBM SPSS Statistics 20 Methylation450 BeadChip Significant CpG sites Choosing a suitable method Primer Design Detection Data analysis

Human IBM SPSS Statistics 20 Methylation450 BeadChip Significant CpG sites Choosing a suitable method Primer Design Detection Data analysis

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