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 Statistical Algorithms Description Document (2002) Affymetrix Inc. Now, this product has been replaced by GeneChip® Operating Software (GCOS).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 1234 5678 91 01 11 2 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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This note was uploaded on 08/26/2009 for the course STAT 416 taught by Professor Peng,l during the Spring '08 term at Iowa State.

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

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