Genomics I:The Transcriptome

RNA Expression AnalysisRNA Expression Analysis

Determining genomewide RNA Determining genomewide RNA expression levelsexpression levels

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

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”

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

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

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.)

The structure of 2’,3’-dideoxynucleotides

Dideoxy Sequencing- The Old Way

The dideoxy sequencing method

Figure 20-16a

© 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458

Summary of chain termination sequencing

Comparison of NextGen Sequencing Platforms

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

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

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

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

Illumina Solexa Strategy

-YouTube animation

-Can generate density of 107 clusters/cm2

SOLiD Sequencing Strategy

ABI site

Ion Torrent Sequencing Strategy

-YouTube animation chemistry