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08%20Normalization%20Affy%202_5_08

08%20Normalization%20Affy%202_5_08 - A Probe Set for a...

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1 1 Normalization and Construction of Expression Measures for Affymetrix GeneChip Data 2/05/2008 Peng Liu 2 A Probe Set for a Particular Gene in GeneChip probe pair probe cell gene sequence ...TGCAATGGGTCAGAA G GACTCCTATGTGCCT... AATGGGTCAGAA G GACTCCTATGTG AATGGGTCAGAA C GACTCCTATGTG perfect match sequence mismatch sequence probe set 3 Affymetrix .CEL Files A .CEL file contains one number representing signal intensity for each probe cell on a single GeneChip. .CEL files can be read with Affymetrix software or in R using the Bioconductor package affy . We will discuss two methods for normalizing and obtaining expression measures using data from Affymetrix .CEL files. 4 Methods 1. Microarray Analysis Suite (MAS) 5.0 Signal proposed by Affymetrix. Statistical Algorithms Description Document (2002) Affymetrix Inc. Now, this product has been replaced by GeneChip® Operating Software (GCOS). GCOS is the same as MAS while working with sample data except that it has a database, networked client-server environment. 2. Robust Multi-array Average (RMA) proposed by Irizarray et al. (2003) Biostatistics 4 , 249-264. These are perhaps the two most popular of many methods for normalizing and computing expression measures using Affymetrix data. Currently over 50 methods are described and compared at http://affycomp.biostat.jhsph.edu/.
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2 5 MAS 5.0 Signal: Background Adjustment Each chip is divided into 16 rectangular zones. The lowest 2% of intensities in each zone are averaged to form a zone-specific background value denoted for zones k=1, 2, ..., 16. The standard deviation of the lowest 2% of intensities in each zone is calculated and denoted for zones k=1, 2, ..., 16. Let denote the distance from the center of zone k to a probe cell located at coordinates (x,y) on the chip. 6 GeneChip Divided into 16 Zones 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 probe cell at coordinates (x,y) x y 7 MAS 5.0 Estimation of ‘local’ background Correction for the background Normalization with mismatch Log-transformation Summarization for each probe set with a robust average (TBW) Scale normalization 8 d 1 (x,y) d 4 (x,y) d 16 (x,y) 16 Distances to Zone Centers for Each Probe Cell
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3 9 MAS 5.0 Signal: Background Adjustment (continued) Let w k (x,y)=1/(d k (x,y)+100). Denote the background for the cell located at coordinates (x,y) by b(x,y)= Denote the “noise” for the cell located at coordinates (x,y) by n(x,y)= Σ k=1 w k (x,y) nZ k / Σ k=1 w k (x,y). 2 16 16 10 MAS 5.0 Signal: Background Adjustment (continued) Let I(x,y) denote the original intensity of the cell located at coordinates (x,y) on the chip. (75 th percentile of 36 pixel intensities in the center of the cell.) Let Define the background-adjusted intensity for the cell at coordinates (x,y) by Henceforth these background-adjusted intensities will be referred to as either PM or MM for perfect match or mismatch cells, respectively.
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