Hello,

my sample sheet looks like this (RNA-seq):

FACTOR  BATCH
factor1 run1
factor1 run2
factor1 run2
factor1 run1
factor2 run1
factor2 run2
factor2 run2
factor2 run1
factor3 run1
factor3 run2
factor3 run2
factor3 run1
factor4 run1
factor4 run2
factor4 run2
factor4 run1

so four conditions over two batches. Batch correction is necessary and beneficial as the effect was clearly visible in a PCA and could be removed with removeBatchEffect from limma.

I use a design of ~ 0 + BATCH + FACTOR as this is what I read in the edgeR manual.

This gives me this matrix:

           run1 run2 FACTOR2 FACTOR3 FACTOR4
1          1         0             0             0             0
2          0         1             0             0             0
3          0         1             0             0             0
4          1         0             0             0             0
5          1         0             1             0             0
6          0         1             1             0             0
7          0         1             1             0             0
8          1         0             1             0             0
9          1         0             0             1             0
10         0         1             0             1             0
11         0         1             0             1             0
12         1         0             0             1             0
13         1         0             0             0             1
14         0         1             0             0             1
15         0         1             0             0             1
16         1         0             0             0             1

I can make most the contrasts I want, e.g. FACTOR2-FACTOR3 via the contrast argument c(0,0,1,-1,0).

Question-1: How would I make FACTOR1-FACTOR2 if I want to use the contrasts rather than the coef argument (because I wrote a script that is based on the contrast argument and I do not want to change it) Is this possible here with contrast? Is it simply c(0,0,-1,0,0)?

Question-2: For one analysis I am interested in the average effect between FACTOR 1 & 2 versus the average of 3 & 4. If I only had the FACTOR level in the matrix with 0+FACTOR I would do (FACTOR1-FACTOR2)/2 - (FACTOR3-FACTOR3)/2 but since FACTOR1 is not part of the matrix, how do I do this here? Should I write 0+FACTOR+BATCH since then all four FACTORs are in the matrix?

Thank you!

I would tend to reverse the order of factor and batch, which makes things easier:

> model.matrix(~0+FACTOR+ BATCH)
   FACTOR1 FACTOR2 FACTOR3 FACTOR4 BATCH2
1        1       0       0       0      0
2        1       0       0       0      1
3        1       0       0       0      1
4        1       0       0       0      0
5        0       1       0       0      0
6        0       1       0       0      1
7        0       1       0       0      1
8        0       1       0       0      0
9        0       0       1       0      0
10       0       0       1       0      1
11       0       0       1       0      1
12       0       0       1       0      0
13       0       0       0       1      0
14       0       0       0       1      1
15       0       0       0       1      1
16       0       0       0       1      0