Genomics I: The Transcriptome RNA Expression Analysis Determining genomewide RNA expression levels.
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Genomics I:The Transcriptome
RNA Expression AnalysisRNA Expression Analysis
Determining genomewide RNA Determining genomewide RNA expression levelsexpression levels
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Genomic analysis of gene expression
• Methods capable of giving a “snapshot” of RNA expression of all genes
• Can be used as diagnostic profile– Example: cancer diagnosis
• Can show how RNA levels change during development, after exposure to stimulus, during cell cycle, etc.
• Provides large amounts of data• Can help us start to understand how whole
systems function
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Basics of microarrays
• DNA attached to solid support– Glass, plastic, or nylon
• RNA is labeled– Usually indirectly
• Bound DNA is the probe– Labeled RNA is the
“target”
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Cell-cycle regulated genes
• Each gene is a line on the longitudinal axis
• Treatments in different panels
• Cell-cycle stages are color coded at top
• Vertical axis groups genes by stage in which expression peaks
Brown and Botstein, 1999
Alpha cdc15 cdc28 Elu
M/G1
G1
S
G2
M
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Analysis of microarray results
• Inherent variability: need for repetition– Biological and technical replicates
• Analysis algorithms– Based on statistical models
• Means of generating hypotheses that need to be tested
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RNA- seq
• High throughput sequencing of cDNAs from biological samples
• Determine abundance of mRNA by representation in sequencing reads
• May detect variants (alternative splicing, specific alleles, etc.)
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The structure of 2’,3’-dideoxynucleotides
Dideoxy Sequencing- The Old Way
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The dideoxy sequencing method
Figure 20-16a
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© 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458
Summary of chain termination sequencing
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Comparison of NextGen Sequencing Platforms
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Pyrosequencing I (454 Strategy)
• Based on production of pyrophosphate during sequencing reaction
• Each time polymerase adds nucleotide (dNTP) to the growing strand, pyrophosphate (PPi) is released– Amount released equal to number of nucleotides
added
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Pyrosequencing II
• To quantitate amount of PPi released:– ATP sulfurylase converts PPi to ATP– ATP used by enzyme luciferase to produce
light from the substrate luciferin• The amount of light produced is directly
proportional to the amount of ATP, which is proportional to the amount of PPi released
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Pyrosequencing III
• Sequential addition of each dNTP gives sequence
• Apyrase enzyme used to degrade dNTPs after reaction completed
• Sequence read from amount of light emitted as each dNTP is added
Nucleotide sequence
Nucleotide added
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Transcriptome Analysis by Pyrosequencing
•Like SAGE in that portions of each RNA are sequenced; abundance proportional to representation in sequencing•“Micro reaction chambers” formed using emulsion of oil and aqueous phases to separate clones•Sequencing strategy different from standard methodology; allows analysis of thousands of clones on one slide•http://www.pyrosequencing.com/DynPage.aspx?id=7454 •http://www.roche-applied-science.com/publications/multimedia/genome_sequencer/amplicon_07/wbt.htm
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Illumina Solexa Strategy
-YouTube animation
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-Can generate density of 107 clusters/cm2
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SOLiD Sequencing Strategy
ABI site
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Ion Torrent Sequencing Strategy
-YouTube animation chemistry
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