Lecture 6: Genomics

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Lecture 6 Genomics

• What is it?
• How is it used?
• What do we do with all this information?
• How is it useful for imaging?
• Terminology genome, genomics, bioinformatics,
  microarray, gene expression profile, DNA chips,
  hybridization, array validation, probe sets, data
  sets

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What Watson and Crick Started
1953 Structure of DNA (Watson and Crick) 1977
DNA sequencing (Sanger) 2001 Human genome
sequenced (Human Genome Project, Celera Genomics)
WHAT is a GENOME?
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• The human genome is the the complete DNA content
  of the 23 pairs of human chromosomes
• - approximately 3.5 billion base pairs.

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The story of two men
Francis Collins Human Genome Project
J. Craig Venter Celera Genomics
www.ornl.gov/sci/techresources/Human_Genome/home.s
html
Levy S, Sutton G, Ng PC, Feuk L, Halpern AL, et
al. (2007). "The Diploid Genome Sequence of an
Individual Human". PLoS Biology 5 (10)
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What did the HGP set out to do?

• identify all the approximately 20,000-25,000
  genes in human DNA,
• determine the sequences of the 3 billion chemical
  base pairs that make up human DNA,
• store this information in databases,
• improve tools for data analysis,
• transfer related technologies to the private
  sector
• address the ethical, legal, and social issues
  (ELSI) that may arise from the project

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What other genomes have been sequenced?

• Mouse (Mus musculus)
• Rat (Rattus novegicus)
• Caenorhabditis elegans
• Drosophila melanogaster
• Bacillus anthracis
• Cow (Bos taurus)
• Rice
• Cotton
• 180 organisms have had their genomes sequenced

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What are we going to do with all this
information??
Discuss.
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Genomics...What Is It?

• An operational definition
• The application of high throughput automated
  technologies to molecular biology.
• A philosophical definition
• A wholistic or systems approach to the study of
  information flow within a cell.

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• Functional Genomics
• Comparative Genomics
• Pharmacogenomics
• Metabolic Genomics (Metabolomics??)

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Gene Expression Analysis
Compare changes in the expression of different
genes between different experimental conditions.
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How do YOU Microarray?
Spotted cDNA Array
Affymetrix Genechip? High-density oligonucleotide
array
Mouse Panc Chip 6 13,000 cDNAs Beta Cell Biology
Consortium www.betacell.org
Mouse Exon 1.0 ST Array One million probes
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Data Analysis.NOW, the Fun Begins!
Bioinformatics
Data Analysis Services dChip GeneSpring GX
v7.3 GeneSpring GT 2 Partek Genomics
Suite Biotique's X-Ray Ingenuity Pathway
Analysis Ariadne Pathway Studio TIGR
MultiExperiment Viewer EASE, HCE, NetAffx

• statistical tools
• gene expression patterns eg clustering
• graphical displays of patterns
• provides meaningful filters to confirm if genes
  have changed, and by how much
• feed data into annontation programs to identify
  genes in terms of function, metabolic pathways,
  sequences

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Online Data Analysis Tools
Power Calculation Gene Cruiser Affy Regional
Bias QC Onto Express NetAffx GO
Mining GenePublisher unCHIP GeneAssist Pathway
Analysis Rat Annotation Tool CyberT
http//www.broad.mit.edu/cancer/genecruiser/
http//www.cbs.dtu.dk/services/GenePublisher/
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Validation of Results

• need to validate your findings
• mRNA transcript levels (qPCR)
• protein expression Western blot,
  immunofluorescent microscopy
• protein function enzyme assay,
  secretion/trafficking analysis, immunofluorescent
  microscopy, electron microscopy

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An Example of the Usefullness of Microarray
Analysis
McGirr R, Ejbick CE, Carter DE, Andrews JD, Nie
Y, Friedman TC, Dhanvantari S 2005 Glucose
dependence of the regulated secretory pathway in
?TC1-6 cells. Endocrinology 1464514-4523

• Observation Glucagon secretion increases during
  onset of Type 1 diabetes (paradoxical
  hyperglucagonemia)
• Question What are the cellular mechanisms that
  cause increased glucagon secretion?
• Method
• Glucagon-secreting cells were treated with
  either low or high glucose for 5 days
• Microarray (low glucose vs. high glucose)
• Validation Western blot and microscopy

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Array Affymetrix GeneChip Mouse Genome
2.0 Analysis GeneSpring 6.1, NetAffx GO Stats
Genes called present in at least one array
ANOVA and Welchs t-test, p
The most interesting genes that changed were
those involved in hormone secretion
Synaptophysin-like protein VAMP4 Syntaxin
1A RAB3D Synaptotagmin 13, 11 PC1/3 proglucagon
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Validation 1 Expression of proteins involved in
secretion
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Validation 2 Immunofluorescence microscopy of
markers for islet differentiation
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• Conclusion
• Chronic exposure to high glucose up-regulates
  cellular secretory machinery
• Provides a mechanism to explain paradoxical
  hyperglucagonemia in diabetes.

Gene Expression Profiling results in the rapid
identification of large numbers of genes that
change in response to different treatments, or
that differ between normal vs disease tissue.
Where's the Imaging?