GCB/CIS 535 Microarray Topics John Tobias November 8th, 2004.

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Presentation transcript:

GCB/CIS 535 Microarray Topics John Tobias November 8th, 2004

Overview Normalization Sample Quality Control Finding Differentially Expressed Genes Gene List Annotation

Overview Normalization Sample Quality Control Finding Differentially Expressed Genes Gene List Annotation

Normalization Adjustment of gene expression values across an experiment Necessary step - corrects systematic biases, facilitates biological comparison Assumptions about sample (and condition) similarities can help focus on true differences

The Box Plot

Linear Scaling Assumption - Median Expression Equal On Each Chip

MedianIQR Assumption - Specific Percentiles Equal On Each Chip

Other Normalizations “Housekeeping” Genes - Assume that expression of some specific genes is unchanging LOESS (LOWESS) - 2-color - Intensity Dependent

Overview Normalization Sample Quality Control Finding Differentially Expressed Genes Gene List Annotation

Sample Quality Control Affymetrix “report” values % Present, 3’/5’ ratios, scaling factor Clustering or PCA for sample similarity Looking for absence of technical grouping – biological condition grouping may be present or absent depending on magnitude of expression changes

PCA for Sample QC

PCA for Sample Comparison

Overview Normalization Sample Quality Control Finding Differentially Expressed Genes Gene List Annotation

Finding Differentially Expressed Genes Choice of algorithm determined by experimental design and goals Multiple testing problem (why p-vaules can be misleading)

Gene List Characteristics Ranked by statistical significance for differential expression Gene 384(high significance) Gene 983 Gene 324 Gene 002 Gene 334 Gene 623 Gene 356 Gene 342 Gene 078 Gene 645 Gene 453 Gene 235 Gene 193 Gene 903 Gene 411 Gene 212 (low significance) True Positives False Positives True Negatives False Negatives

Data for a Single Gene ControlTreated Expression X X X X X X

Pool Genes to Estimate Error ControlTreated

Data for a Single Gene ControlTreated Expression X X X X X X

Comparing Gene Lists

Differential Expression Algorithms Affymetrix Microarray Suite v5 Significance Analysis of Microarrays (SAM) Local Pooled Error (LPE) NIA Website (choice of variance models) Mixed Model Multi-way ANOVA

Overview Normalization Sample Quality Control Finding Differentially Expressed Genes Gene List Annotation

Gene List Annotation Pathways Functional Groups Affymetrix GeneSpring DAVID GoMiner GenMapp

Identifiers to Knowledge

Ingenuity Pathway Analysis Curated Interaction and Pathway Database Mine literature as it relates to gene list

Contact Information Penn Bioinformatics Core - 13th Floor Blockley Hall John Tobias Reserve Computers