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

▶
principles and challenges of dna methylation profiling
SMART_READER_LITE
LIVE PREVIEW

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

Apr 09, 2023 428 likes •783 views

Principles and challenges of DNA methylation profiling Department of Forensic Medicine Yonsei University College of Medicine Epigenetics DNA methylation DNA methylation is an epigenetic mechanism used by cells to control gene

slide-1
SLIDE 1

Principles and challenges of DNA methylation profiling

Department of Forensic Medicine Yonsei University College of Medicine

정 상 은

slide-2
SLIDE 2

Epigenetics

slide-3
SLIDE 3

DNA methylation

 DNA methylation is an epigenetic mechanism used by cells to control gene expression.

slide-4
SLIDE 4

A suitable method for DNA methylation analysis

slide-5
SLIDE 5

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

DNA Methylation Analysis Method

slide-6
SLIDE 6

DNA Methylation Analysis (Bisulfite conversion)

slide-7
SLIDE 7

Human Methylation 450 BeadChip

slide-8
SLIDE 8
  • 8 -

Human Methylation 450 Provides coverage.

slide-9
SLIDE 9

Human Methylation 450 Workflow

slide-10
SLIDE 10

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

slide-11
SLIDE 11

Pyrosequencing

slide-12
SLIDE 12

MSP (Methylation Specific PCR/qPCR)

slide-13
SLIDE 13

Capillary Electrophoresis (CE) System

slide-14
SLIDE 14

NGS : Whole-Genome Bisulfite Sequencing

slide-15
SLIDE 15

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
slide-16
SLIDE 16

Human Methylation450 BeadChip Significant CpG sites Choosing a suitable method Primer Design Detection Data analysis

slide-17
SLIDE 17

Human Methylation450 BeadChip Significant CpG sites Choosing a suitable method Primer Design Detection Data analysis < Body Fluid Identification>

slide-18
SLIDE 18

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

slide-19
SLIDE 19

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
slide-20
SLIDE 20

Human Methylation450 BeadChip Significant CpG sites Choosing a suitable method Primer Design Detection Data analysis http://genome.ucsc.edu/index.html

slide-21
SLIDE 21

Human Methylation450 BeadChip Significant CpG sites Choosing a suitable method Primer Design Detection Data analysis http://genome.ucsc.edu/cgi-bin/hgBlat

slide-22
SLIDE 22

Human Methylation450 BeadChip Significant CpG sites Choosing a suitable method Primer Design Detection Data analysis

slide-23
SLIDE 23

Human Methylation450 BeadChip Significant CpG sites Choosing a suitable method Primer Design Detection Data analysis

slide-24
SLIDE 24

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

slide-25
SLIDE 25

Human Methylation450 BeadChip Significant CpG sites Choosing a suitable method Primer Design Detection Data analysis

slide-26
SLIDE 26

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

slide-27
SLIDE 27

Human Methylation450 BeadChip Significant CpG sites Choosing a suitable method Primer Design Detection Data analysis

slide-28
SLIDE 28

Human Methylation450 BeadChip Significant CpG sites Choosing a suitable method Primer Design Detection Data analysis Bisulfite converted sequence (CG  YG, C  T)

slide-29
SLIDE 29

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

slide-30
SLIDE 30

Human Methylation450 BeadChip Significant CpG sites Choosing a suitable method Primer Design Detection Data analysis

slide-31
SLIDE 31

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

slide-32
SLIDE 32

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

slide-33
SLIDE 33

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

slide-34
SLIDE 34

Tha Thank nk y you fo for y your ur a atte ttenti ntion! n!