Complete Sample to Analysis Solutions for DNA Methylation ......CAG EMEAI | Agilent Restricted |...

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Complete Sample to Analysis Solutions

for DNA Methylation Discovery

using Next Generation Sequencing

SureSelect Human/Mouse Methyl-Seq

Kyeong Jeong PhD

February 5, 2013


DNA Methylation: Background

DNA Methylation: Enzymatic modification of

cytosine in CG dinucleotides

• Maintained through cell division

• Both DNA strands are methylated

• Platform for Methyl binding proteins

• Protein recruitment leading to compact, silent

chromatin unavailable for transcription initiation

• Gene silencing, imprinting, X-inactivation, tissue

specific repression

• Genome Stability


Common targets for DNA methylation

Differentially methylated regions (DMR)

• CpG islands (e.g. 4~8 % tissue-specific differentially methylated regions or T-DMR)

• CpG island shores (~2kb away from islands, e.g. 76% of T-DMRs in shores)

Irizarry RA et al. Nature Genetics 2009

HS3ST4 :

heparan sulfate D-glucosaminyl 3-O-

sulfotransferase 4


Role in cancer

• Hypomethylation in heterochromatin: Genomic instability

• Hypermethylation in tumor suppressor gene: Transcriptional repression of TSG

DNA methylation: Significance

Robertson K. Nature Genetics, 2005


DNA methylation: Significance

Role in other diseases

• Neurodevelopmental disorders

– X-linked α-thalassemia and metal retardation (ATRX syndrome)

– Fragile X syndrome

– ICF (Immune deficiency, Centromeric instability, and Facial abnormalities)

• Imprinting disorders

– Prader-Willi syndrome

– Angelman syndrome

– Beckwith-Wiedemann syndrome


Current methods

o NGS-based assay

• MethylC-Seq: Whole genome shotgun sequencing with bisulfite-treated DNA (1 bp)

• RRBS (Reduced Representation Bisulfite Sequencing):

Methylation-insensitive restriction enzyme & Bisulfite treatment (1 bp)

• MeDIP-Seq: Antibody for methylated DNA (150 bp)

• MBD-Seq: Methyl-binding domain protein (150 bp)

• MRE-seq: Restriction enzyme to detect unmethylated DNA (1 bp)

o Microarray assay

• Single base methylation assay (450k or 27k)

• CHARM (Comprehensive High-throughput Arrays of Relative Methylation)

• Antibody-based assay

LIMITATIONS

Cost of WGS

Bias from enzyme / antibody

Lack of single base pair

resolution

Content limitations


SureSelectXT Human Methyl-Seq

CONTENT - 84 Mb Design, 3.7M CpGs

CpG islands

Cancer, Tissue-specific DMRs

GENCODE promoters

Known DMRs or Regulatory features in

•Shores and shelves ±4kb

•DNAse hypersensitive sites

•Under-methlated regions

•RefSeq Genes

•Ensemble Regulatory Features

•Reduced bias – Other methods use

enzymes or antibodies that can bias

towards specific sequences or

methylation states.

•Discovery Tool - Probes are not

methylation state dependent so you do

not need to have prior knowledge of the

methylation states of the regions that

you want to target

•Comprehensive design - Not limited

to CpG Islands. Comprehensive

targeting key methylation regions:

CpG Islands, Promoters and DMRs

•High sensitivity - Ability to distinguish

individual CpG sites


SureSelectXT Human Methyl-Seq (84 Mb Design, 3.7M

CpGs)

Site Classification Covered regions (bp) CpGs covered by baits

CpG Islands

(~91% of UCSC annotated CpG islands) 19,605,556 1,679,870

Cancer-, Tissue-Specific DMRs

(~23,000 DMRs; Most are from Irizarry RA et al. Nat. Genet.

2009 Feb;41(2):178-86)

9,773,047 293,619

Gencode promoters

(~141,000 promoters; 1kb-upsteam from TSS;

All genes in Gencode v7 are included except repeat regions)

36,974,007 1,272,026

~482,000 DMRs or regulatory features in

- CpG Island shores/shelves (±4 kb)

- Enhancers

- Ensemble regulatory regions

- Dnase I hypersensitive sites

48,021,626 2,057,280


SureSelectXT Mouse Methyl-Seq

Site Classification Number of Targets Total Bases Covered

CpG Islands 16,027 10,512,276 bp

Tissue-specific DMRs 33,456 10,452,692 bp

Ensembl Regulatory

Features - CpG shores and shelves

(±4kb)

- DNase I Hypersensitive sites

- Histone Modifications

- TFBS

- Polymerase

171,796 91,799,015 bp

Open Regulatory Annotation

(ORegAnno) - Promoters

- Enhancers

- TFBS

- Regulatory Polymorphisms

14,951 9,983,957 bp

Provided by Dr. Druley (Washington Univ.) / 109Mb / Early Access


SureSelect Target Enrichment

3,200,000,000 bp

84,000,000 bp 38x efficiency =

Whole genome

vs. SureSelect

• Focus on regions of Methylation significance


SureSelect Target Enrichment

DNA

Shearing End repair ‘A’ tailing

Capture/

Wash

Hybridization

(24hr)

PCR

Sequencing

mAdapter

ligation

Bisulfite

treatment

A A

me me me me

me me me me

A A

Index PCR

Bismark

Alignment

Me Me Sodium

Bisulfite


Methyl-Seq analysis Workflow

Bisulfite

treatment

Sequencing

Demultiplexing

Alignment

% Methylation

Computation

QC

Capture

performance

Summary

Pre

pro

cessin

g


Capture performance

8Gb of sequencing

Percentage reads in targeted regions 86.0%

Percentage reads in regions +/- 100bp: 95.3%

Percent of genome targeted: 2.7%

Percentage of targeted bases covered by at least 1 read

98.2%


94.8%


90.0%


Whole genome data vs. SureSelect Methyl-Seq

R = 0.93 R = 0.99

Whole genome bisulfite sequencing data: Lister R. et al. 2009

(IMR90: Fetal lung fibroblasts)

http://www.chem.agilent.com/Library/posters/Public/AGBT_MethylSeq_poster_Feb2012.pdf

-Tissue Specific DMRs

http://www.chem.agilent.com/Library/posters/Public/AGBT_MethylSeq_poster_Feb2012.pdf


SureSelect Methyl-Seq vs. Illumina 450K array

R=0.96


Methyl-Seq

Illumina 450K

Colon cancer cells (HCT116 vs. Methyltransferase DKO)

HCT116 (Colon cancer cell line)

HCT116 DKO: Methyltransferase double knockout (DNMT1-/- & DNMT3b-/-)

HCT116 HCT116 DKO

Highly sensitive and accurate detection of DNA methylation changes


Methylation at single base-pair resolution

(GNAS: G-protein alpha subunit )

HCT116

SureSelect

HCT116DKO

SureSelect

HCT116

450K

HCT116DKO

450K

Minimize missing

information More confidence

on subtle changes

Detect gradual

changes

DMR

? ? ?


Applications for Non-CpG methylation

• Stem cells

– Lister R. et al. 2009 Human DNA methylomes at base resolution show

widespread epigenomic differences. Nature

• Mouse Genome

– Xie W. et al. 2012 Base-Resolution Analyses of Sequence and Parent-of-Origin

Dependent DNA Methylation in the Mouse Genome. Cell

• Nucleosome positioning / Chromatin Accessibility

– Kelly T. K. et al. 2012 Genome-wide mapping of nucleosome positioning

and DNA methylation within individual DNA molecules, Genome Research


Conclusions

•SureSelect Methyl-Seq Target Enrichment Platform:

• Comprehensive

• Robust

• Cost-effective

•SureSelect Methyl-Seq allows for single base-pair resolution.

•Excellent concordance with published whole genome data.

•Content is focused most on important regions of the human

methylome.


Methylation

Effects on Gene

Expression RNA DNA

SureSelect: Complete “Omics” Solution

DNA: Genetic variation

RNA: Gene Expression

Methyl: Effects on Gene Expression


Acknowledgements

University of Washington:

o John Stamatoyannopoulos

- Tony Shafer

- Eric Haugen

University of California San Diego:

o Kun Zhang

Johns Hopkins University:

o Andy Feinberg

o Sarven Sarbuncian

Washington University:

o Todd Druley


Thank You!

Complete Sample to Analysis Solutions for DNA Methylation ......CAG EMEAI | Agilent Restricted |...

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