Question: Affymetrix Data Using Mas5 Giving Large Fc Values

0

Bade •

**40**wrote:HI All,

I am new to Bioconductor and using affy+limma packages to analyze affymetrix data. Everything works well when I use RMA method in affy expresso but when I use MAS5 than in results I get values which are way to high for fold change (results below).

My Code:

```
data.mas <- expresso(data,bgcorrect.method="mas",normalize.method="qspline",pmcorrect.method="mas",summary.method="mas")
eset <- exprs(data.mas)
design <- model.matrix(~ 0+factor(c(1,1,1,2,2,2,3,3,3,4,4,4,)))
colnames(design) <- c("A.10.5", "B.11.5", "C.12.5","XYZ")
fit <- lmFit(eset, design)
contrast.matrix <- makeContrasts(A.10.5-WB,B.11.5-WB,C.12.5-WB,levels=design)
fit2 <- contrasts.fit(fit, contrast.matrix)
fit2 <- eBayes(fit2)
results <- (topTable(fit2, coef=1, adjust='fdr',sort.by='T', p.value = 1, n=30))
```

Results:

```
ID logFC AveExpr t P.Value adj.P.Val
19419 1435113_x_at -1031.5628 328.50510 -75.54197 7.176278e-14 2.657009e-09
9834 1425528_at -755.6372 291.45928 -71.46655 1.178248e-13 2.657009e-09
7586 1423280_at -939.7628 305.33928 -67.55115 1.949728e-13 2.931156e-09
7912 1423606_at -941.8785 309.39784 -59.27310 6.270564e-13 7.070218e-09
40160 1455865_at -293.2026 100.87426 -48.26481 3.926156e-12 3.541471e-08
```

But when do preprocessing using RMA (code below) instead of MAS5 I get correct log FC values:

```
data.rma <- expresso(data,bgcorrect.method="rma",normalize.method="quantiles",pmcorrect.method="pmonly",summary.method="medianpolish")
eset <- exprs(data.rma)
```

Could you plese tell me what am I missing?

Thanks

AK