Introduction to Affymetrix Introduction to Affymetrix MicroarraysMicroarrays

Why use microarrays?Permits expression profiling of thousands of genes in parallel

Why use Affymetrix microarrays?• Well established platform• Arrays for various species• Enables combination of different applications • Support from the bioinformatic community• Well annoted probesets

Introduction to microarraysIntroduction to microarraysSteps in analysisSteps in analysis

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DesignDesign ExperimentExperiment

DesignDesign ExperimentExperiment

ProbeProbeArrayArray

ProbeProbeArrayArray

PreparePrepareSampleSample

PreparePrepareSampleSample

Wash&

Stain

FluidicsStation

Hybridize

Hybridization Oven

Data Analysis Software

Introduction to microarraysIntroduction to microarraysAffymetrix InstrumentationAffymetrix Instrumentation

5 µm

Thousands of copies of a specificoligonucleotide probe

Image of Hybridized GeneChip Array

>6.5 million differentcomplementary probes

Single stranded, labeled ‘target’

Oligonucleotide ‘probe’

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*****GeneChip® Array Hybridized Probe Cell

1.28cm

1.28cm

Introduction to microarraysIntroduction to microarraysChip features Chip features

Copy Number ChangesCopy Number Changes

Differential Gene ExpressionDifferential Gene Expression

Splice Variant AnalysisSplice Variant Analysis

Somatic MutationsSomatic Mutations

Mitochondrial MutationsMitochondrial Mutations

•• WG SNP Mapping ArraysWG SNP Mapping Arrays

•• Exon ArraysExon Arrays•• 33’’ IVT Expression ArraysIVT Expression Arrays

•• Exon ArraysExon Arrays

•• Whole Genome Tiling ArraysWhole Genome Tiling Arrays•• Promoter Tiling ArraysPromoter Tiling Arrays

•• Mito Resequencing Mito Resequencing

•• Targeted Genotyping ArraysTargeted Genotyping Arrays•• CustomSeq Resequencing ArraysCustomSeq Resequencing Arrays

GenotypingGenotyping•• WG SNP Mapping ArraysWG SNP Mapping Arrays•• Targeted Genotyping ArraysTargeted Genotyping Arrays

Changes in Changes in MethylationMethylation PatternsPatterns

Introduction to microarraysIntroduction to microarraysArrays and ApplicationsArrays and Applications

Introduction to microarraysIntroduction to microarraysAffymetrix ApplicationsAffymetrix Applications

Introduction to microarraysIntroduction to microarraysDevelopment of Affymetrix technologyDevelopment of Affymetrix technology

Experimental Planning - Variance estimation - Level of significance - How many replicates?

Low-level analysis - Background correction - Normalization - Summarization

Quality Control - GCOS internal controls - Various methods in R / BioConductor

Statistical methods - Pairwise comparisons - Significance testing (t-test, SAM etc) - Clustering - Classification & Prediction

Functional Analysis - Pathways - GeneOntologies - Diseases - Motifs - Toxicogenomics - Pharmacogenomics ….

Introduction to microarraysIntroduction to microarraysBioinformatic flowchartBioinformatic flowchart

Lab tasks:• Download data set from GEO

• Add annotations from Affymetrix

• Perform t-tests to compare expression between groups

• Answer a set of questions using web resources

Primary Recurrentstage IIIB or IV

Adenocarcinoma (AC)

10 15

Squamous cell carcinoma (SCC)

10 8

Title: Molecular profiling of tumor progression in NSCLC

Geo id: GSE7880

Array: Affymetrix Human HG-Focus Target Array

Design:

Computer Lab Computer Lab --in silicoin silico microarray study microarray study

http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE7880

Computer LabComputer LabQuality Control of dataQuality Control of data