introduction to affymetrix microarrays why use microarrays? permits expression profiling of...
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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
Scan
Scanner
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