06%20LOWESS%20Normalization%201_29_08

06%20LOWESS%20Normalization%201_29_08 - Within-slide...

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1 1 LOWESS Normalization for Two-Color Microarray Data 1/29/2008 Peng Liu 2 Notations: ± For a spot j, j=1,…,N, (N is the total number of spots on a slide), let R j and G j denote the background corrected intensities for the red and green dyes respectively. ± R j and G j = (SMD j –BMD j ) for red and green respectively, if we use local background correction. ± Log intensity ratio: M j = log(R j ) – log(G j ) ± Average log intensity: A j = [log(R j ) + log(G j )]/2 3 Within-slide normalization ± This is done separately for each slide. ± The purpose is to make red intensities and green intensities comparable. ± It is known that for self-self experiments (for which the same sample is labeled with Cy3 and Cy5), the green intensities tend to be higher than the red intensities. And the difference is not constant across spots. 4 Figure 2 from Dudoit et al, 2002, Statistica Sinica
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2 5 Dye effect ± Possible reasons : Physical properties of dyes (heat and light sensitivity, half life) Efficiency of dye incorporation Scan settings (laser voltage setting, photon emission response to laser excitation) etc. ± Result: between two channels on the same slide 6 A real data example Log Red Log Green 7 Normalization: M vs. A Plot (45 o rotation) - Log Green = M ( Log Green + Log Red )/2 = A 8 LOWESS normalization ± LOWESS stands for ± The original reference for lowess is Cleveland, W. S. (1979). Robust locally weighted regression and smoothing scatterplots. JASA 74 829- 836. ± It was recommended to correct the intensity related dye bias by Yang, et al. ( 2002. Nucleic Acids Research, 30, 4 e15 ) and Dudoit et al, 2002, Statistica Sinica.
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3 9 LOWESS Fit ( Log Green + Log Red )/2 Log Red - Log Green 10 After normalization A Normalized M 11 Assigned Readings ± Yang, et al. (2002). Normalization for cDNA microarray data: a robust composite method addressing single and multiple slide systematic variation, Nucleic Acids Research , 30, 4 e15 ± Handout 12 LOWESS “LOcally WEighted polynomial regreSSion” ± The process is considered
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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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06%20LOWESS%20Normalization%201_29_08 - Within-slide...

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