DNA methylation_ understanding the language of DNA 20130806

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Advances in Epigenetics

Swati Kadam, Ph.D. Marketing Intern

Aug 2013

Cumulative papers

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

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What is Epigenetics?

http://www.cashou.com/portfolio/human_body/index.html#

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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.


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Lets eat, Grandpa!

Lets eat Grandpa!

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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 site CpG 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

,

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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

Introduction: Importance of DNA methylation

Calico Cats: Female calicos have a splotched coat

pattern linked to X-inactivation.

DNA methylation (Full-Stop)

Females

14

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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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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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

+ =

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

Techniques currently used to map 5mC and 5hmC

Differences between the techniques

Summary of workflows

Mapping Epigenetic Modifications on DNA

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


Full Stop Comma


DNA methylation (5mC)

DNMTs

DNA Methyl

Transferase

TETs

Ten-Eleven

Translocation

1/2/3

+

5mC 5hmC


DNA demethylation

TETs

Ten-Eleven

Translocation

1/2/3

+ Other Proteins


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



RRBS-Seq: Reduced Representation BiSulfite Sequencing

MspI digestion

Genomic DNA

Illumina

Sequencing

Illumina

methylated

adapters

BiSulfite

Conversion

Size

Selection

PCR

amplification



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


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


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


Full Stop Comma


DNA hydroxymethylation (5hmC)

DNMTs

DNA Methyl

Transferase

TETs

Ten-Eleven

Translocation

1/2/3

+

5mC 5hmC


DNA demethylation

TETs

Ten-Eleven

Translocation

1/2/3

+ Other Proteins

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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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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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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

31

5hmC methods summary

Reduced Representation Hydroxymethyl Profiling Sequencing (RRHP-seq)

HydroxyMethylated – DNA Immunoprecipitation

(HMe-DIP)

Tet-Assisted Bisulfite Sequencing

(TAB-Seq)

Library

Prep

+

+

32

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.

34

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

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Reference Guides:

Focal Points


Importance of Epigenetic modifications

Current methods used to map modifications

Workflows for each method

Where Illumina fits into the picture


- 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?

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Questions about presentation:

– Swati Kadam, Product Marketing Intern

[email protected]

– Abizar Lakdawalla, Product Management [email protected]

For more information about WGBS kit:

– Fraz Syed, Sr. Product Marketing

[email protected]

Contact Information:

mailto:[email protected]

DNA methylation_ understanding the language of DNA 20130806

Health & Medicine

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