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<strong>Outline</strong>• What is Low-Level Analysis?• Affymetrix GeneChip Technology• Two topics in Low-level analysis– Constructing a gene expression measure– Probe level models for detecting differentialexpression
Low-Level Analysis• What is low-level analysis?– Analysis and manipulation of probe intensity data• Expression calculation: Background, Normalization,Summarization• Determining presence/absence• Quality control diagnostics• Why do we do it?– Hopefully it will allow us to produce better, morebiologically meaningful gene expression values– We want accurate (low bias) and precise (lowvariance) gene expression estimates– Is there additional information at the probe-levelthat we might otherwise throw away?
Page 4 and 5: High-Level Analysis• Clustering/C
Page 6 and 7: Brief TechnologyOverview• High de
Page 8 and 9: Two Probe TypesPM: the Perfect Matc
Page 10 and 11: Focusing on a SingleGeneChip Cell L
Page 12 and 13: Hybridization to the Chip
Page 14 and 15: Chip dat file - checkered board - o
Page 16 and 17: Chip dat file - checkered board - c
Page 18 and 19: Constructing a geneexpression measu
Page 20 and 21: Background/SignalAdjustment• A me
Page 22 and 23: Normalization“Non-biological fact
Page 24 and 25: Summarization• Reduce the 11-20 p
Page 26 and 27: Affymetrix Spike-in Data• 59 chip
Page 28 and 29: Assessing Bias:Observed Expression
Page 30: Assessing Variability:M vs A plots
Page 38 and 39: Summary of Trade-offsBackgroundMeth
Page 43 and 44: Scaling is Not Sufficient
Page 45 and 46: Little effect on Spike-insMethodAll
Page 47 and 48: Comparing EstablishedExpression Mea
Page 49 and 50: General Probe LevelModelyij= f( X)+
Page 51 and 52: Fitting the PLM• Robust regressio
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Fold ChangeFC = X − XlmWhereXl=
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“Robust” t-statistict=Xlsnm2s2l
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Limma “ebayes” t-statistic• G
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Probe Level Model teststatisticstPL
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What Happens as the Numberof Arrays
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ResultsMethod Individual Models Sin
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What is going on here?• Examine r
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Low Non-Differential
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High Non-Differential
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GeneLogic Tonsil dataset
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ResultsMethod 3 vs 3 4 vs 4 5 vs 50
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Ongoing work in this area• Techno
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Additional Slides
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Original RMA Background• Convolut
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A Standard Curve AdjustmentBased on
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γRelates to Concentration
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The Two Curves Yield anAdjustment C
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Sort columns of of originalmatrixTa
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RMA Model• To each probeset (k),
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Basic RMA modelLetthenyij= log N B2
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SlopeAllMidLowHighValue0.4930.6650.
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SlopeAllMidLowHighValue0.5890.7510.
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SlopeAllMidLowHighValue0.6950.820.5
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Slope: 0.484
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Slope: 0.583
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Slope: 0.692
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