07 Microarray Microarray technology Data acquisition Image processing Data analysis alessandro bogliolo isti information science and technology institute 1/17 Microarray technology Array Type Spot Density (per cm 2 ) Probe Target Labeling Nylon Macroarrays < 100 cdna RNA Radio Nylon Microarrays < 5000 cdna mrna Radio/Flour Glass Microarrays < 10,000 cdna mrna Fluor Oligonucleotide Chips <250,000 oligo's mrna Fluor alessandro bogliolo isti information science and technology institute 2/17
Microarray experiment 30min 1hr 2hr 4hr 30min 2MA 1hr 2MA 2hr 2MA 4hr 2MA 30min AIG 1hr AIG 2hr AIG 4hr AIG. 132 conditions Gene 1 Gene 2 Gene 3 Gene 4.. 5281 genes alessandro bogliolo isti information science and technology institute 3/17 Microarray scanner alessandro bogliolo isti information science and technology institute 4/17
Microarray scanner alessandro bogliolo isti information science and technology institute 5/17 Noise Noise sources: Sample preparation, labeling, amplification Reaction variations Environment Target volume Hybridization parameters (temperature, time,...) Aspecific hybridization Dust Scanner settings Quantization alessandro bogliolo isti information science and technology institute 6/17
Noise filtering (1) Gridding: identify spot locations Segmentation: distinguish foreground from background Fixed Circle: put a circle around the foreground area Seeded region growing: identify initial spot seeds and grow high intensity regions Edge detection algorithms Background cancellation Intensity = FGintensity - BGintensity alessandro bogliolo isti information science and technology institute 7/17 Noise filtering (2) alessandro bogliolo isti information science and technology institute 8/17
Quality evaluation Irregular size or shape Irregular placement Low intensity Saturation Spot variance Background variance indistinguishable bad print saturated misalignment artifact alessandro bogliolo isti information science and technology institute 9/17 Normalization (1) Normalize data to correct for artificial variances Red = FGred - BGred Green = FGgreen BGgreen PixelValue = log 2 (Red/Green)- log 2 (Red avg /Green avg ) Pixel color: Green if pixel value < 0 Yellow if pixel value = 0 Red if pixel value > 0 alessandro bogliolo isti information science and technology institute 10/17
Normalization (2) Uncalibrated, red light under detected Calibrated, red and green equally detected alessandro bogliolo isti information science and technology institute 11/17 Data analysis: Scatterplot alessandro bogliolo isti information science and technology institute 12/17
Data analysis: Classification Goal: Identify subset of genes that distinguish between treatments, tissues, etc. Method Collect several samples grouped by treatments (e.g. ALL vs. AML) Use genes as features Build a classifier to distinguish treatments Classifiers Neural networks, decision trees,... alessandro bogliolo isti information science and technology institute 13/17 Principal component analysis alessandro bogliolo isti information science and technology institute 14/17
Clustering Hypothesis: Genes with similar function have similar expression profiles Find groups of genes with similar expression profiles Find groups of individuals with similar expression profiles within a population alessandro bogliolo isti information science and technology institute 15/17 Clustering: k-mean algorithm A. B. C. D. alessandro bogliolo isti information science and technology institute 16/17
Hyerarchical clustering Top-down (division) Bottom-up (agglomeration) alessandro bogliolo isti information science and technology institute 17/17