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Back to the Basics:
Methyl-Seq 101
Presented By:
Alex Siebold, Ph.D.
October 9, 2013 Field Applications Scientist
Agilent Technologies
Life Sciences & Diagnostics Group
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Event Date & Time Speaker Topics
NGS Data Analysis 101 Thu, Oct 10
1 pm ET
Jean Jasinski, PhD
Field Application
Scientist
• Analysis Workflows, File Formats,
and Data Filtering
• DNA-Seq vs. RNA-Seq
Considerations
• Integrating Disparate Data Sets to
Create a More Complete Story
NGS Panels 101 Fri, Oct 11
1 pm ET
Adam Hauge,
University of
Minnesota
• Panel Design Process
• Quality at the Bench: Tips, Tricks,
and Lessons Learned
• Considerations for Future Panels
Back to the Basics: Agilent’s Five Part 101
eSeminar Series
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CAG EMEAI | Agilent Restricted | Page 3 Life Sciences & Diagnostics Group | Agilent Technologies | Page 3 S1
Topics for Today’s Presentation
Technology Behind SureSelectXT 2
1
3 SureSelectXT Human Methyl-seq
4
Epigenetics & DNA Methylation
Comparing DNA Methylation Methods
5
Not approved for use in diagnostic
procedures
GeneSpring & Additional TE Solutions
6 Summary & Upcoming 101 eSeminars
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Defining Epigenetics & Epigenetic Protein
Functions • Epigenetics: Studies changes in gene expression caused by mechanisms
that do not effect the underlying DNA sequence
- ex: DNA Methylation and Covalent Modification of Histone Tails
DNMT1 (DNA Methyltransferse)
MLL (Histone Methyltransferse)
Methyl CpG Binding Proteins (MBD’s)
CBX7 (Chromodomain)
BRD4 (Bromodomain)
DNA Demethylation
HDAC’s (Histone deacetylases)
UTX (Histone demethylase)
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DNA Methylation: An Epigenetic Modification
• Found in animals, plants, bacteria, fungi, etc…
• Initially thought to be a static modification, but is dynamic
• Important in Development and Cellular Differentiation
• Promotes Gene Silencing
• Aberrant DNA methylation contributes to a host of diseases,
including cancer
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CpG Dinucleotides & Their Genomic
Locations
CpG islands
• High frequency of CpG dinucleotides
– > 500bp & GC content >55% & observed/expected CpG ratio > 0.65
• In or near about 40% of promoters of mammalian genes
Promoters
• 75% of transcriptional start sites have CpG-rich regions
• 88% of active promoters are associated with CpG-rich sequences
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Differentially Methylated Regions (DMRs)
• CpG islands
– 4~8 % tissue-specific differentially methylated regions or T-DMR
• CpG island shores
– ~2kb away from islands, 76% of T-DMRs in shores
• CpG island shelves
– ~4 kb away from islands
Irizarry RA et al. Nature Genetics 2009
HS3ST4 :
heparan sulfate D-
glucosaminyl 3-O-
sulfotransferase 4
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The Study of Epigenetics & How it Relates to
the Clinic
•DNA Methylation has a rapidly growing
role in cancers and other diseases
•Pharma & Biotech are actively seeking
new small molecules that inhibit certain
classes of epigenetic enzymes
FDA Approved Epigenetic Therapies
Vidaza & Dacogen: Inhibit DNA Methyltransferases
Vorinostat & Romidepsin: Inhibit Histone Deacetylases
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Impact of Adding Methylation Assays to Your
Current Research
• Better understand the link between environment and
genetics
• Obtain the complete picture of a gene and its impact on the
pathogenesis of cancer or other disease state
9 Not approved for
use in diagnostic
procedures
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Topics for Today’s Presentation
Technology Behind SureSelectXT 2
1
3 SureSelectXT Human Methyl-seq
4
Epigenetics & DNA Methylation
Comparing DNA Methylation Methods
5
Not approved for use in diagnostic
procedures
GeneSpring & Additional TE Solutions
6 Summary & Upcoming 101 eSeminars
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The Power Behind the Performance Agilent’s Core Competency: Oligo Library Synthesis (OLS)
HP Inkjet Printer Technology + Proprietary Chemistry =
Long High-Quality Oligos (SurePrint Technology)
Agilent’s SureSelect Platform Utilizes 120bp Oligos
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Target Enrichment: It’s just like fishing…
Why perform target enrichment?
1. Sequence only your desired regions of
interest (Exons, gene panels, intergenic
regions etc...)!
2. Sequence more samples per lane/run
(i.e. Multiplex)
3. Save time and money
4. Faster time to results = Smaller datasets
5. Identify variants in samples with increased
reliability and accuracy:
More Reads in regions of interest =
Higher Depth of Coverage
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SureSelect: In-Solution Target Enrichment S
ure
Sele
ct D
NA
Nat Biotechnol. 2009 Feb;27(2):182-9.
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SureSelect: In-Solution Target Enrichment S
ure
Sele
ct D
NA
Nat Biotechnol. 2009 Feb;27(2):182-9.
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Target Enrichment Maximizes Your
Sequencing Efficiency x Desired Depth of Coverage = Required Seq Depth/Sample
Human Genome
3Gb x 30 = 90Gb
Illumina HiSeq 2000 37Gb/lane
~ 3 lanes per sample!
$$$$$
Illumina HiSeq FlowCell
Genome Size
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Target Enrichment Maximizes Your
Sequencing Efficiency x Desired Depth of Coverage = Required Seq Depth/Sample
Human Genome
3Gb x 30 = 90Gb
Illumina HiSeq 2000 37Gb/lane
Illumina HiSeq FlowCell
Target Size
v
Target = 50Mb x 100 = 5Gb
Target = 5Mb x 100 = 500Mb
Target = 500Kb x 100 = 50Mb
Target = 50Kb x 100 = 5Mb
Develop designs/panels for
any sequencing capacity:
- High Throughput or Desktop
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CAG EMEAI | Agilent Restricted | Page 17 Life Sciences & Diagnostics Group | Agilent Technologies | Page 17 S1
Topics for Today’s Presentation
Technology Behind SureSelectXT 2
1
3 SureSelectXT Human Methyl-seq
4
Epigenetics & DNA Methylation
Comparing DNA Methylation Methods
5
Not approved for use in diagnostic
procedures
GeneSpring & Additional TE Solutions
6 Summary & Upcoming 101 eSeminars
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SureSelectXT Human Methyl-Seq
Discovery Tool
• Not methylation-state dependent
• No prior knowledge needed
Comprehensive design
• Not limited to CpG Islands
• Comprehensive Content
• CpG Islands, Promoters and
DMRs
DESIGN CONTENT - 84 Mb Design,
3.7M CpGs
CpG islands
Cancer, Tissue-specific DMRs
GENCODe promoters
DMRs or regulatory features in:
CpG Islands, shores and shelves ±4kb
DNAseI hypersensitive sites
Refseq Genes
Ensembl Regulatory Features
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SureSelectXT Human Methyl-Seq Bait design
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SureSelectXT Methyl-Seq Workflow
DNA
Shearing, End
Repair & ‘A’
addition
SureSelect
Hyb (24hr)
mAdapter
ligation
Bisulfite
treatment
me me me me
me me me me
A A
Library
Quant and
PCR
Sequence
Bisulfite treatment is
performed after
hybridization to
maximize sample
complexity
No PCR before
Bisulfite treatment
to preserve the
Methylation state
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Highly sensitive and accurate methylation detection after SureSelect target enrichment
demonstrated DNA methylation differences between HCT116 human colon cancer cells
and its methyltransferase double-knockout (DNMT1-/- and DNMT3b-/-).
Proof of Concept: HCT116 vs Methyltransferase DKO
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Proof of Concept: Methylation of Metalloproteinase inhibitor 3
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Proof of Concept:
Identifying Tissue Specific DMRs
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SureSelectXT Methyl-Seq Capture
Performance
Percentage reads in targeted regions: 82.0%
Percentage reads in regions +/- 100bp: 93.6%
Percent of genome targeted: 2.7%
Enrichment in targeted regions: 30.07
Uniformity (3/4 mean with upper tail): 91.4%
Number of bases in targeted regions: 84,367,621
Percentage of targeted bases covered by...
...at least 1 read: 98.7%
...at least 10 reads: 91.4%
...at least 20 reads: 78.9%
10Gb Sequencing per Sample
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CAG EMEAI | Agilent Restricted | Page 25 Life Sciences & Diagnostics Group | Agilent Technologies | Page 25 S1
Topics for Today’s Presentation
Technology Behind SureSelectXT 2
1
3 SureSelectXT Human Methyl-seq
4
Epigenetics & DNA Methylation
Comparing DNA Methylation Methods
5
Not approved for use in diagnostic
procedures
GeneSpring & Additional TE Solutions
6 Summary & Upcoming 101 eSeminars
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Comparative Publication Of Methyl Assays:
Nature Biotech. (28), pp: 1026–1028: (2010)
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Whole Genome Bisulfite Sequencing (WGBS)
• Whole Genome Coverage
• But…costly and time consuming
• Requires extensive bioinformatics
• Limited scalability per run
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MeDip-Seq & Reduced Representation
BiSulfite Sequencing (RRBS)
• Difficult to target specific regions (i.e. DMRs in Shelf and Shore regions)
• Biased towards methylated regions, Repeat sequences & CpG-rich
sequences
• Can miss under-methylated regions
• Difficult to design since knowledge of methylation state for the target
region is needed
Limitations
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Microarray-Based Method: Infinium 450K
Array
• Improved cost and throughput vs. whole-genome bisulfite sequencing
• Not whole-genome (~480K Features)
• Does not cover shores and shelves known to house important DMRs
• CpG Rich Regions are hard to resolve at single base resolution
• What are you missing?
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SureSelectXT Methyl-Seq
• What if you could: • reduce cost/sample
• Interrogate regions more
efficiently than WGBS
• Confidently identify both
Hyper & Hypo methylated
regions in a single experiment
• Have increased scalability
• Achieve high depth of coverage
to confidently make methyl calls
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Methyl-Seq Comparison with published
WGBS data
Cell line: IMR90 (female lung fibroblast)
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SureSelect Methyl-seq vs. Whole-Genome
Bisulfite Sequencing
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Concordance between WGBS data &
SureSelect Methyl-Seq
R = 0.927
Excellent concordance with whole genome bisulfite sequencing
data.
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Excellent Reproducibility
Excellent reproducibility for IMR90 Replicate 1 vs. 2
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SureSelectXT – Complete Workflow Solution
SureSelectXT Human Methyl-Seq
Target Enrich. Library
Prep
Indexes
35
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CAG EMEAI | Agilent Restricted | Page 36 Life Sciences & Diagnostics Group | Agilent Technologies | Page 36 S1
Topics for Today’s Presentation
Technology Behind SureSelectXT 2
1
3 SureSelectXT Human Methyl-seq
4
Epigenetics & DNA Methylation
Comparing DNA Methylation Methods
5
Not approved for use in diagnostic
procedures
GeneSpring & Additional TE Solutions
6 Summary & Upcoming 101 eSeminars
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Integrated Biology Applications in GeneSpring
Including Methyl-Seq Analysis!
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Integrated Biology Applications in GeneSpring
Including Methyl-Seq Analysis!
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• Each Kit has a unique ELID identifying number
• All kit design files can be easily retrieved from Agilent SureDesign
• Access SureDesign to create your own custom captures for FREE!
Sure
Sele
ct D
NA
& R
NA
Additional Target Enrichment Solutions:
SureSelectXT
Targets Kits Species Capture
Exomes 50Mb (V3) V4, V4+UTR
V5, V5+UTR (NEW!)
Human, Mouse,
Zebrafish, bovine,
dog
DNA-Seq
DNA
Methylation
Human Methyl-Seq (84Mb)
Mouse Methyl-Seq
(100Mb)
Human
Mouse (EA)
DNA-Seq
Kinome DNA or RNA ~3 Mb Human DNA or RNA-Seq
Custom
Regions
>0.2 Mb to 34 Mb
Any DNA or RNA-Seq
Focused
Panels
>0.2 Mb to 34 Mb
Any DNA or RNA-Seq
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CAG EMEAI | Agilent Restricted | Page 40 Life Sciences & Diagnostics Group | Agilent Technologies | Page 40 S1
Topics for Today’s Presentation
Technology Behind SureSelectXT 2
1
3 SureSelectXT Human Methyl-seq
4
Epigenetics & DNA Methylation
Comparing DNA Methylation Methods
5
Not approved for use in diagnostic
procedures
GeneSpring & Additional TE Solutions
6 Summary & Upcoming 101 eSeminars
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SureSelectXT Human Methyl-Seq Summary
• Covers more individual relevant CpGs compared to
Methylation microarrays
• Reveals methylated regions undetected by RRBS and
Me-Dip
• Increases throughput, reduces costs compared to
WGBS and allows better usage of sequence capacity
• Produces higher quality data than WGBS, deeper reads,
faster analysis
• Identification of DMR in relevant genes can lead to
discovery of useful biomarkers
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A Beginner’s Glossary for Methyl-seq:
Walk the walk, talk the talk
1. Epigenetics – The study of changes in gene expression that are caused by mechanisms that do not effect the underlying
DNA sequence. Examples include covalent modification to histones tails and the methylation of DNA.
2. Epigenetics Writers – Individual enzymes or protein complexes that facilitate the establishment of covalent modifications to
DNA or histones. Examples include DNA methyltransferase and histone methyltransferase.
3. Epigenetic Readers - Proteins that identify specific epigenetic marks and either directly bind to or recruit proteins to bind to
them in order to modulate gene expression. Examples include methyl CpG binding proteins or members of the Polycomb and
Trithorax group proteins.
4. Epigenetic Erasers – Proteins that can remove covalent modifications to DNA and histones.
5. CpGs – Regions of the genome where cytosines precede guanines along the linear DNA sequence. The “p” in the CpG
annotation stands for phosphate which means the cytosine nucleotide occurs 5’ of the guanine nucleotide. This nomenclature
is used to prevent confusion since cytosines form Watson-Crick base pairing with guanines, which are not sites for DNA
methylation.
6. CpG Islands – Regions of the genome, typically >500bp, that contain a high density of CpG dinucleotide sequences.
7. CpG Island Shores – Term that describing the regions of differentially methylated CpG dinucleotides which occur
approximately 2 kb away from annotated CpG islands .
8. CpG Island Shelves – Similar to CpG shores, however these regions are found even further from annotated CpG islands in
the genome, approximately 4 kb away from annotated CpG islands.
9. DMRs – Referring to Differentially Methylated Regions of the genome.
Not approved for use in diagnostic
procedures
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Agilent Technologies Knows NGS 101 & More…
Offering Complete Solutions for NGS Workflows
The Gold Standard for Sample QC
2100 Bioanalyzer Instrument & Kits
2200 TapeStation Instrument & Kits
NGS Analysis Software
GeneSpring NGS
SureCall
Validation Technologies
qPCR- Mx system & Brilliant reagents
Microarrays- CGH, CGH+SNP,
Gene Expression & miRNA
The Leader in NGS Target Enrichment
SureDesign
SureSelect
HaloPlex
Bravo Automation
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Event Date & Time Speaker Topics
NGS Data Analysis 101 Thu, Oct 10
1 pm ET
Jean Jasinski, PhD
Field Application
Scientist
• Analysis Workflows, File Formats,
and Data Filtering
• DNA-Seq vs. RNA-Seq
Considerations
• Integrating Disparate Data Sets to
Create a More Complete Story
NGS Panels 101 Fri, Oct 11
1 pm ET
Adam Hauge,
University of
Minnesota
• Panel Design Process
• Quality at the Bench: Tips, Tricks,
and Lessons Learned
• Considerations for Future Panels
Back to the Basics: Agilent’s Five Part 101
eSeminar Series Continues…
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Contact Us
800.227.9770
[email protected]
www.agilent.com/genomics