12 Maart 2013

Slides available www.bioinformatics.be

Lab for Bioinformatics and computational genomics

10 “genome hackers” mostly engineers (statistics)

42 scientiststechnicians, geneticists, clinicians

>100 people hardware engineers,

mathematicians, molecular biologists

Can bioinformatics bridge the gap ?

The genome is just the start …

Epigenetic (meta)information = stem cells

250 different cell types

Epigenetic (meta)information = stem cells

Cellular programming

Defining Epigenetics

Reversible changes in gene expression/function

Without changes in DNA sequence

Can be inherited from precursor cells

Allows to integrate intrinsic with environmental signals (including diet)

Genome

DNA

Gene Expression

Epigenome

Chromatin

Phenotype

DNA Methylation Differentiates Totipotent Embryonic Stem Cells from Unipotent Adult Stem Cells

Alex Meissner, Henry Stewart Talks

Paula Vertino, Henry Stewart Talks

Reprogramming the DNA methylome

Transgenerational inheritence

The epigenome is actionable

The epigenome is actionable

Epigenetic Changes are Important in Causing Cancer

Example: Replication errors

GENETIC EPIGENETIC

Example: Chromatin modification errors

Altered DNA/mRNA/proteins

Altered DNA sequence

Altered levels ofmRNA/proteins

Alteredchromatin structure

X X

Oncogenesis

Tumor

Source: Schuebel et al 2007

Methylated Mutated

76-100 51-75 21-50 1-20

Dx

CDx

Example of Methylation vs Mutation: Colon & Breast Cancer

MGMT BiologyO6 Methyl-Guanine Methyl Transferase

Essential DNA Repair Enzyme

Removes alkyl groups from damaged guanine bases

Healthy individual: - MGMT is an essential DNA repair enzymeLoss of MGMT activity makes individuals susceptible to DNA damage and prone to tumor development

Glioblastoma patient on alkylator chemotherapy: - Patients with MGMT promoter methylation show have longer PFS and OS with the use of alkylating agents as chemotherapy

MGMT Promoter Methylation Predicts Benefit form DNA-Alkylating Chemotherapy

Post-hoc subgroup analysis of Temozolomide Clinical trial with primary glioblastoma patients show benefit for patients with MGMT promoter methylation

Median Overall Survival

21.7 months

12.7 months

radiotherapy

plus temozolomide

Methylated MGMT Gene

Non-Methylated MGMT Gene

radiotherapy

Adapted from Hegi et al.NEJM 2005352(10):1036-8.Study with 207 patients

# samples

# markers

Profiling the Epigenome

Discovery

Verification

Validation

Genome-wide methylation by methylation sensitive restriction enzymes

Genome-wide methylation by probes

# samples

# markers

Profiling the EpigenomeBy next gen sequencing

Discovery

Verification

Validation

MBD_Seq

DNA Sheared

Immobilized Methyl Binding Domain

Condensed Chromatin

DNA Sheared

Immobilized Methyl binding domain

MgCl2

Next Gen SequencingGA Illumina: 100 million reads

MBD_Seq

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Kit Comparison

MBD_SeqMGMT = dual core

# samples

# markers

MBD_Seq

Profiling the epigenome…. by next generation sequencing

Discovery

1-2 millionmethylation

cores

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Bock et al. Nature 2012Bock et al, Nature, 2012

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Data integrationCorrelation tracks

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methylation methylation

expression expression

Corr =-1 Corr = 1

Correlation trackin GBM @ MGMT

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+1

-1

miRNA, (l)ncRNA, CIS/TRANS splicing, SV, fusion loci , bidirectional promoters ?

RNA_seq: sequence RNA molecules Next Gen Platform

Total RNA_seq: all RNA molecules (normalisation procedure)

Directional Total RNA_seq: before amplification use different 5’ and 3’ adaptors

Integrated Directional Total RNA_seq: Combine with other datasets eg. enrichment sequencing data, visualise and query in genome browser

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Next_next

Direction RNAseq bidirectional promoters

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# samples

# markers

MBD_Seq

454_BT_Seq

Profiling the Epigenome…. by next generation sequencing

Discovery

Verification

Validation

GCATCGTGACTAGCGACTGATCGATGGATGCTAGCAT

Where is the mC ?

GCATCGTGACTAGCGACTGATCGATGGATGCTAGCAT

GCATCGTGACTAGCGACTGATCGATGGATGCTAGCAT

25% 50% 25%

GCATCGTGACTAGCGACTGATCGATGGATGCTAGCAT

GCATCGTGACTAGCGACTGATCGATGGATGCTAGCAT

25% 50% 25%

GCATCGTGACTAGCGACTGATCGATGGATGCTAGCAT

GCATCGTGACTAGCGACTGATCGATGGATGCTAGCAT

Dense methylated needed for transcriptional silencingAre there alleles with all three positions methylated ?

GCATCGTGACTTACGACTGATCGATGGATGCTAGCAT

unmethylated alleles

less methylationmethylated alleles

more methylation

Deep Sequencing

Deep MGMTHeterogenic complexity

Combination of different sequencing techniques is emerging as best practiceBioinformatics is challengingMethods for normalisation under constructionReference databases are generated Data visualization and integration is key

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Conclusion

4th December 2012Johns HopkinsBloombergSchool of Public Health

Slides available www.bioinformatics.be

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