Studies with more than two groups

Differential Expression Analysis with limma in R

John Blischak

Instructor

A study with 3 groups

3 different types of leukemias: ALL, AML, CML
- Bioconductor package: leukemiasEset
- Kohlmann et al. 2008, Haferlach et al. 2010

dim(eset)

Features  Samples
   20172       36

table(pData(eset)[, "type"])

ALL AML CML
 12  12  12

Group-means model for 3 groups

$$ Y = \beta_1 X_1 + \beta_2 X_2 + \beta_3 X_3 + \epsilon $$

$\beta_1$ - Mean expression level in group ALL
$\beta_2$ - Mean expression level in group AML
$\beta_3$ - Mean expression level in group CML
Tests:
- AML v. ALL: $\beta_2 - \beta_1 = 0$
- CML v. ALL: $\beta_3 - \beta_1 = 0$
- CML v. AML: $\beta_3 - \beta_2 = 0$

Group-means design matrix for 3 groups

design <- model.matrix(~0 + type, 
                       data = pData(eset))

head(design, 3)

          typeALL typeAML typeCML
sample_01       1       0       0
sample_02       1       0       0
sample_03       1       0       0

colSums(design)

typeALL typeAML typeCML
     12      12      12

Contrasts matrix for 3 groups

AML v. ALL: $\beta_2 - \beta_1 = 0$
CML v. ALL: $\beta_3 - \beta_1 = 0$
CML v. AML: $\beta_3 - \beta_2 = 0$

library(limma)
cm <- makeContrasts(AMLvALL = typeAML - typeALL,
                    CMLvALL = typeCML - typeALL,
                    CMLvAML = typeCML - typeAML,
                    levels = design)

cm

         Contrasts
Levels    AMLvALL CMLvALL CMLvAML
  typeALL      -1      -1       0
  typeAML       1       0      -1
  typeCML       0       1       1

Testing 3 groups

library(limma)
# Fit coefficients
fit <- lmFit(eset, design)

# Fit contrasts
fit2 <- contrasts.fit(fit, contrasts = cm)

# Calculate t-statistics
fit2 <- eBayes(fit2)

# Summarize results
results <- decideTests(fit2)
summary(results)

   AMLvALL CMLvALL CMLvAML
-1     898    3401    1890
0    18323   13194   16408
1      951    3577    1874

The effect of hypoxia on stem cell function

3 different levels of oxygen: 1%, 5%, 21%
- Bioconductor package: stemHypoxia
- Prado-Lopez et al. 2010

dim(eset)

Features  Samples
   15325        6

table(pData(eset)[, "oxygen"])

ox01 ox05 ox21
   2    2    2

Let's practice!

Differential Expression Analysis with limma in R