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
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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
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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
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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
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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
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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
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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
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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
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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
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SureSelect Target Enrichment
3,200,000,000 bp
84,000,000 bp 38x efficiency =
Whole genome
vs. SureSelect
• Focus on regions of Methylation significance
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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
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Methyl-Seq analysis Workflow
Bisulfite
treatment
Sequencing
Demultiplexing
Alignment
% Methylation
Computation
QC
Capture
performance
Summary
Pre
pro
cessin
g
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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%
Percentage of targeted bases covered by at least 5 read
94.8%
Percentage of targeted bases covered by at least 10 read
90.0%
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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
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SureSelect Methyl-Seq vs. Illumina 450K array
R=0.96
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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
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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
? ? ?
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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
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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.
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Methylation
Effects on Gene
Expression RNA DNA
SureSelect: Complete “Omics” Solution
DNA: Genetic variation
RNA: Gene Expression
Methyl: Effects on Gene Expression
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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
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Thank You!