A review of next generation DNA sequencing based methods for characterization of DNA methylation and hydroxymethylation. Presentation by Illumina.

1. © 2013 Illumina, Inc. All rights reserved.
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of Illumina, Inc. All other brands and names contained herein are the property of their respective owners.
Advances in Epigenetics
Swati Kadam, Ph.D.
Marketing Intern
Aug 2013

2. Cumulative papers

3. Focal Points
• What is Epigenetics?
• Importance of Epigenetic modifications
• Current methods used to map modifications
• Workflows for each method
• Where Illumina fits into the picture

4. 4
What is Epigenetics?
http://www.cashou.com/portfolio/human_body/index.html#

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What is Epigenetics?
http://www.cashou.com/portfolio/human_body/index.html#
Total number of cells in the body ~50 trillion:
50,000,000,000,000 cells
– Each with it’s own instruction manual (DNA)
 ~3164.7 million bases (A, T, G, C)
 ~2% of the human genome codes for protein
 ~20,687 total genes (~3000 bases each)

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Epigenetics: Study of heritable changes in gene
expression that occur without changes in the primary
DNA sequence.
What is Epigenetics?

7. 7
Lets eat, Grandpa!
Lets eat Grandpa!

8. 8
Epigenetic modifications are like punctuation
marks on the DNA

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Zaidi S K et al. Mol. Cell. Biol. 2010;30:4758-4766
Introduction: Epigenetic Regulation

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• Methylation of Cytosines: 5mC
• Hydroxymethylation of Cytosines: 5hmC
• Cytosines next to Guanines: CpG sites
• Many CpGs together: CpG island
• Most located near TSS
• CpG Shores are sites 2 kilo bases away from CpG Islands
Introduction: Epigenetic Lingo
CpG Island CpG siteCpG Shores 5mC 5hmC

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• DNMT: DNA MethylTransferases
• DNMT1, DNMT3A/3B, DNMT3L
• Maintain DNA methylation mark
• TET: Ten-Eleven-Translocation
• TET1, TET2, TET3
• Play an important role in removing
methylC (5mC) to form
hydroxymethylC (5hmC) (an
intermediate in demethylation).
Introduction: Epigenetic Lingo
,

12. 12
Full Stop Comma
Pfeifer, G. P., S. Kadam, et al. (2013). "5-hydroxymethylcytosine and its potential roles in development and cancer." Epigenetics Chromatin 6(1): 10.
DNA methylation (5mC) and DNA hydroxymethylation (5hmC)
DNMTs
DNA Methyl
Transferase
TETs
Ten-Eleven
Translocation
1/2/3
+
5mC 5hmC
Proposed intermediate to
DNA demethylation
TETs
Ten-Eleven
Translocation
1/2/3
+
Other Proteins

13. 13
Introduction: Importance of DNA methylation
Calico Cats: Female calicos have a splotched coat
pattern linked to X-inactivation.
DNA methylation (Full-Stop)
Females

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Introduction: How are 5mC or 5hmC modifications relevant?
Iqbal, K., Jin, S. G., Pfeifer, G. P. & Szabo, P. E. Reprogramming of the paternal genome upon fertilization involves genome-wide oxidation of 5-methylcytosine. Proc. Natl
Acad. Sci. USA 108, 3642–3647 (2011)
Mom-5mC Dad-5hmC

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Introduction: How are 5mC or 5hmC modifications relevant?
X
Mutations in DNMT1: embryo dies
Mutations in DNMT 3B: Genetic disorder
Immunodeficiency-centromeric instability- Facial
anomalies syndrome (ICF)X
Brown KD, Robertson KD. DNMT1 knockout delivers a strong blow to genome stability and cell viability. Nat Genet. 2007;39(3):289–290.

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Introduction: How are 5mC or 5hmC modifications relevant?
Koh K. P., Yabuuchi A., Rao S., Huang Y., Cunniff K., Nardone J., et al. (2011). Tet1 and Tet2 regulate 5-hydroxymethylcytosine production and cell lineage specification in
mouse embryonic stem cells. Cell Stem Cell 8, 200–213. doi: 10.1016/j.stem.2011.01.008.
Dawlaty M. M., Breiling A., Le T., Raddatz G., Barrasa M. I., Cheng A. W., et al. (2013). Combined deficiency of Tet1 and Tet2 causes epigenetic abnormalities but is compatible
with postnatal development. Dev. Cell 24, 310–323. doi: 10.1016/j.devcel.2012.12.015.
X
Mutations in Tet1: viable fetus
Mutations in Tet1+ Tet2: death after birth
Mutated in Blood Cancers

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 Epigenetic modifications are punctuation marks on DNA
– DNA methylation (5mC) on Cytosine  FULL STOP
– DNA hydroxymethylation (5hmC) on Cytosine  COMMA
 Important during growth for proper formation of organs and adult cells
 Imbalance in 5mC or 5hmC leads to cancer
Take Home Message 1: Epigenetic Regulation
+ =

18. Focal Points
• What is Epigenetics?
• Importance of Epigenetic modifications
• Current methods used to map modifications
• Workflows for each method
• Where Illumina fits into the picture

19. 19
 Techniques currently used to map 5mC and 5hmC
 Differences between the techniques
 Summary of workflows
Mapping Epigenetic Modifications on DNA

20. 20 COMPANY CONFIDENTIAL – INTERNAL USE ONLY
Human: 5hmC
Monocytes: ~0.06% of cytosines
moDCs: ~0.10% of cytosines
moMOs: ~0.11% of cytosines
Human: 5mC
Monocytes: ~4.8% of cytosines
moDCs: ~4.6% of cytosines
moMOs: ~4.5% of cytosines
These modifications are like finding a needle in a haystack
Mouse: 5mC
~1% of bases
~5% of cytosines
~6 x 107 in ES cells
Mouse: 5hmC
~0.1% of bases
~0.5% of cytosines
~6 x 106 in ES cells

21. 21 COMPANY CONFIDENTIAL – INTERNAL USE ONLY
Full Stop Comma
Pfeifer, G. P., S. Kadam, et al. (2013). "5-hydroxymethylcytosine and its potential roles in development and cancer." Epigenetics Chromatin 6(1): 10.
DNA methylation (5mC)
DNMTs
DNA Methyl
Transferase
TETs
Ten-Eleven
Translocation
1/2/3
+
5mC 5hmC
Proposed intermediate to
DNA demethylation
TETs
Ten-Eleven
Translocation
1/2/3
+
Other Proteins

22. 22 COMPANY CONFIDENTIAL – INTERNAL USE ONLY
5mC mapping technique
C
G
G
C
C
G
G
C
Bisulfite Conversion
C
G
G
C
C
G
G
C
C
G
G
C
C
G
G
C
U
G
G
U
U
G
G
U
BS-Seq: BiSulfite Sequencing
Library Prep
+
Genome wide
sequencing
Methylated UnMethylated
C
G
G
C
C
G
G
C
T
G
G
T
T
G
G
T

23. 23 COMPANY CONFIDENTIAL – INTERNAL USE ONLY
5mC mapping technique
RRBS-Seq: Reduced Representation BiSulfite Sequencing
MspI digestion
Genomic DNA
Illumina
Sequencing
Illumina
methylated
adapters
BiSulfite
Conversion
Size
Selection
PCR
amplification

24. 24 COMPANY CONFIDENTIAL – INTERNAL USE ONLY
5mC mapping technique
Me-DIP-Seq: Methylated DNA ImmunoPrecipitation Sequencing
Fragmentation
Size Selection
Adapter Ligation
Denature to
ssDNA
5mC antibody
Illumina
Sequencing
PCR
amplification
Enrichment of
5mC DNA

25. 25 COMPANY CONFIDENTIAL – INTERNAL USE ONLY
5mC mapping technique - methyl group
- GST-MBD2b
- His-MBD3L1
Rauch, T.A., Li, H., Wu, X., Pfeifer, G.P., (2006). “MIRA-assisted microarray analysis, a new technology for the determination of DNA methylation patterns, identifies
frequent methylation of homeodomain-containing genes in lung cancer cells.” Cancer Res. 66: (7939-7947)
MIRA-Seq: Methylated-CpG Island Recovery Assay Sequencing
Fragmentation
Size Selection
Adapter Ligation
Incubation
MIRA enrichment
of the 5mC DNA
Genomic DNA
Isolate GST-
MBD2b/DNA
complex
Illumina
Sequencing
GST-bead

26. 26 COMPANY CONFIDENTIAL – INTERNAL USE ONLY
Reduced Restriction
BiSulfite
(RRBS-Seq)
Methylated – DNA
Immunoprecipitation
(Me-DIP)
BiSulfite Sequencing
(BS-Seq)
Library
Prep
+
Methylated CpG Island
Recovery Assay (MIRA)
Bis-SNP
Bismark
Bison
BSMAP
MethylCoder
DNAA
5mC methods summary

27. 27 COMPANY CONFIDENTIAL – INTERNAL USE ONLY
Full Stop Comma
Pfeifer, G. P., S. Kadam, et al. (2013). "5-hydroxymethylcytosine and its potential roles in development and cancer." Epigenetics Chromatin 6(1): 10.
DNA hydroxymethylation (5hmC)
DNMTs
DNA Methyl
Transferase
TETs
Ten-Eleven
Translocation
1/2/3
+
5mC 5hmC
Proposed intermediate to
DNA demethylation
TETs
Ten-Eleven
Translocation
1/2/3
+
Other Proteins

28. 28
5hmC mapping technique
Yu, M., G. C. Hon, et al. (2012). "Tet-assisted bisulfite sequencing of 5-hydroxymethylcytosine." Nat Protoc 7(12): 2159-2170.
TAB-Seq: Tet-Assisted Bisulfite Sequencing

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5hmC mapping technique
RRHP-Seq: Reduced Representation Hydroxymethylcytosine Profiling Sequencing
MspI digestion
Genomic DNA
Adapters
hmC
protection
by
glucosylation
5hmC
5mC
MspI
digestion
Illumina
Sequencing
PCR
amplification
5hmC
5mC
Size selection of
enriched
5hmC DNA

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5hmC mapping technique
HMe-DIP-Seq: HydroxyMethylated DNA ImmunoPrecipitation Sequencing
Fragmentation Adapters
Denature to
ssDNA
5hmC antibody
5hmC
5mC
Genomic DNA
Illumina
Sequencing
PCR
amplification
Size selection of
enriched
5hmC DNA

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5hmC methods summary
Reduced Representation
Hydroxymethyl Profiling
Sequencing (RRHP-seq)
HydroxyMethylated – DNA
Immunoprecipitation
(HMe-DIP)
Tet-Assisted Bisulfite
Sequencing
(TAB-Seq)
Library
Prep
+
+

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Reduced Representation
Hydroxymethyl Profiling
Sequencing (RRHP-seq)
Tet-Assisted Bisulfite
Sequencing
(TAB-Seq) +
+
HydroxyMethylated – DNA
Immunoprecipitation
(HMe-DIP)
Library
Prep
Reduced Restriction
BiSulfite
(RRBS-Seq)
Methylated – DNA
Immunoprecipitation
(Me-DIP)
BiSulfite Sequencing
(BS-Seq)
+
Methylated CpG Island
Recovery Assay (MIRA)
TruSeq Kits
Nextera Kits
illumina: Total Solution
MiSeq
RRBS-Seq/RRHP-Seq
MeDIP/hMe-DIP
MIRA
HiSeq
WGBS-Seq/TAB-Seq
RRBS-Seq/RRHP-Seq
MeDIP/hMe-DIP
MIRA

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Summary for mapping cytosine modifications
CpG Island CpG site 5mC 5hmC
Modification Method (example) Reagents kit Pre-prep input gDNA
Cost to prepare
DNA
Coverage needed
(Gbp)
Total Cost per sample
5mC
WGBS Zymo EZ DNA methylation-Lightning kit 0.5-2ug ~ $4 90 ~ $5000
RRBS Msp1 Enzyme (NEB) + Zymo EZ DNA methylation-Lightning kit 1-2ug ~ $3 3 ~ $300
Me-DIP MeDIP kit (Active Motif) 0.1-1ug ~ $40 5 ~ $300
MIRA/MBD Active Motif Methyl Collector Ultra kit (Active Motif) 0.01-1ug ~ $20 5 ~ $300
HM 450 array Illumina Infinium Array (Targeted) 0.5ug - - $240
5hmC
TAB WiseGene TAB kit + Zymo EZ DNA methylation-Lightning kit 0.5ug ~ $100 90 ~ $5000
RRHP Zymo Research 3-5 ~ $300
Hme-DIP Hme-DIP kit (Active Motif) 0.1-1ug ~ $40 3-5 ~ $300
Note: An example of the kits is provided, other kits are also substitutable. Please contact manufacturer for details or recommendations
Note: Values presented here are an estimate and are subject to change.

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Technique Pros Cons Areas Covered Areas Missed
BS  Genome wide coverage
of non-CpG methylation.
 Genome wide coverage
of CpGs
 Single -CpG resolution
 GC content
 Sequence complexity
 Difficulty aligning BS reads
to reference.
 Difficulty differentiated
repeat region locations in
genome
 Disadvantageous at C  T
SNPs
 High cost per CpG covered
genome-wide
 Cannot distinguish between
5mC and 5hmC
 CpG Islands dense
 CpG Islands less
dense
 CpGs in repeats
 Non-CpG methylation
 Genome-wide CpG
me
 None
RRBS  Genome wide coverage
of CpGs in Islands
 Low Cost per sample
 Single CpG Resolution
 Msp1 cuts at specific sites
 Measures 10-15% CpGs in
genome
 Dependent on Sequence
 Cannot distinguish between
5mC and 5hmC
 CpG Islands dense  CpG in repeats
 Non CpG me
 Genome-wide CpG
me
 CpG Islands w/o
enzyme site
MeDIP  Genome wide coverage
of non-CpG methylation.
 Genome wide coverage
of CpGs
 Independent of
sequence.
 Lowest cost per CpG
genome-wide
 Does not interact with
5hmC
 Low resolution (~150bp)
 Non-specific interaction
 Bias towards hyper
methylated regions
 CpG Islands dense
 CpG Islands less
dense
 CpGs in repeats
 Non-CpG methylation
 Genome-wide CpG
me
 None
MIRA/MBD  Genome wide coverage
of CpGs in dense CpG
areas
 Does not interact with
5hmC
 Genome wide coverage of
non-CpG methylation.
 Less coverage of low CpG
areas
 Low resolution (~150bp)
 Bias towards
hypermethylated regions
 CpG Islands dense
 CpGs in repeats
 Non CpG me
 CpG Islands less
dense
 Genome wide CpG
me

35. 35
Reference Guides:

36. Focal Points
 What is Epigenetics?
 Importance of Epigenetic modifications
 Current methods used to map modifications
 Workflows for each method
 Where Illumina fits into the picture

37. 37 COMPANY CONFIDENTIAL – INTERNAL USE ONLY
- Identification of novel biomarkers in cancer
- OPT gene 100% methylated in primary breast tumors
- Kim, Myung Soon, et al. "Genome-wide identification of OTP gene as a novel methylation marker of breast cancer."
Oncology reports 27.5 (2012): 1681-1688.
- Identification of changes in location of 5mC and 5hmC in development
- CH methylation accumulates during brain development [non-CpG
methylation]
- Lister, Ryan, et al. "Global Epigenomic Reconfiguration During Mammalian Brain Development." Science (New
York, NY) (2013).
- Changes in epigenome upon treatment with carcinogens
- Non-genotoxic carcinogens causes changes in epigenome. 5hmC
associated with active chromatin state.
- Thomson JP, Lempiainen H, Hackett JA, et al. Non-genotoxic carcinogen exposure induces defined changes in the
5-hydroxymethylome. Genome Biol. 2012;13:R93.
- Monozyotic twins studied at epigenome level
- Same genome, different epigenome Where DNA methylation correlated with
Autism
- Wong CC, Meaburn EL, Ronald A, et al. Methylomic analysis of monozygotic twins discordant for autism spectrum
disorder and related behavioural traits. Mol Psychiatry. 2013 Apr 23. doi: 10.1038/mp.2013.41.
We are just beginning to scratch the surface
=

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Won’t you want to learn the
punctuated language of your
DNA to unravel the mysteries
of life and disease?

39. 39
Questions about presentation:
– Swati Kadam, Product Marketing Intern
 skadam@illumina.com
– Abizar Lakdawalla, Product Management
 alakdawalla@illumina.com
For more information about WGBS kit:
– Fraz Syed, Sr. Product Marketing
 fraz.syed@epicentre.com
Contact Information: