Hello,

I am a new user of the package NanoStringDiff. I am working on an experiment with 4 groups and I am trying to figure out how to design the contrast vector, specifically which numbers to use for each group. To the best of my understanding, "0" omits the group, and the numbers, "1,-1" are used in the examples of the package. Should I use "-1/2, 1/2" for the other 2 groups? Is there any chance that 1/2 takes the average of the corresponding group? In other words, how to decide which numbers to use for the contrast vector if we have more than 2 groups?

Thank you everyone

Assuming you have fit a cell means model (e.g., you used model.matrix(~ 0 + <group factor goes here>)), you should then use 0 for groups you don't want to compare, 1 for the first group and -1 for the second group in your comparison. To make your life easier, you should also use makeContrasts from the limma package.

> design <- model.matrix(~0 + gl(4, 3))
> colnames(design) <- LETTERS[1:4]
> contrast <- makeContrasts(A - B, B - C, C - D, levels = design)
> design
   A B C D
1  1 0 0 0
2  1 0 0 0
3  1 0 0 0
4  0 1 0 0
5  0 1 0 0
6  0 1 0 0
7  0 0 1 0
8  0 0 1 0
9  0 0 1 0
10 0 0 0 1
11 0 0 0 1
12 0 0 0 1
attr(,"assign")
[1] 1 1 1 1
attr(,"contrasts")
attr(,"contrasts")$`gl(4, 3)`
[1] "contr.treatment"

> contrast
      Contrasts
Levels A - B B - C C - D
     A     1     0     0
     B    -1     1     0
     C     0    -1     1
     D     0     0    -1

## A versus B

rslt <- glm.LRT(NanoStringData, design, contrast[,1])

## B versus C
rslt <- glm.LRT(NanoStringData, design, contrast[,2])

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