**0**wrote:

I have a paired design, two groups, two time points and two conditions. Participants are nested within groups. Time points are nested within conditions.

Here it is:

```
group ind.n when time
A 1 A A
A 1 A B
A 1 A C
B 1 A A
B 1 A B
B 1 A C
A 2 A A
A 2 A B
A 2 A C
A 3 A A
A 3 A B
A 3 A C
A 4 A A
A 4 A B
A 4 A C
B 2 A A
B 2 A B
B 2 A C
B 3 A A
B 3 A B
B 3 A C
A 5 A A
A 5 A B
A 5 A C
A 6 A A
A 6 A B
A 6 A C
A 7 A A
A 7 A B
A 7 A C
A 8 A A
A 8 A B
A 8 A C
A 9 A A
A 9 A B
A 9 A C
B 4 A A
B 4 A B
B 4 A C
A 1 B A
A 1 B B
A 1 B C
B 1 B A
B 1 B B
B 1 B C
A 2 B A
A 2 B B
A 2 B C
A 3 B A
A 3 B B
A 3 B C
A 4 B A
A 4 B B
A 4 B C
B 2 B A
B 2 B B
B 2 B C
B 3 B A
B 3 B B
B 3 B C
A 5 B A
A 5 B B
A 5 B C
A 6 B A
A 6 B B
A 6 B C
A 7 B A
A 7 B B
A 7 B C
A 8 B A
A 8 B B
A 8 B C
A 9 B A
A 9 B B
A 9 B C
B 4 B A
B 4 B B
B 4 B C
A 10 A A
A 10 A B
A 10 A C
A 11 A A
A 11 A B
A 11 A C
A 12 A A
A 12 A B
A 12 A C
A 13 A A
A 13 A B
A 13 A C
A 14 A A
A 14 A B
A 14 A C
A 15 A A
A 15 A B
A 15 A C
A 16 A A
A 16 A B
A 16 A C
A 17 A A
A 17 A B
A 17 A C
A 18 A A
A 18 A B
A 18 A C
A 19 A A
A 19 A B
A 20 A A
A 20 A B
A 20 A C
A 21 A A
A 21 A B
A 21 A C
B 5 A A
B 5 A B
B 5 A C
A 10 B A
A 10 B B
A 10 B C
A 11 B A
A 11 B B
A 11 B C
A 12 B A
A 12 B B
A 12 B C
A 13 B A
A 13 B B
A 13 B C
A 14 B A
A 14 B B
A 14 B C
A 15 B A
A 15 B B
A 15 B C
A 16 B A
A 16 B B
A 16 B C
A 17 B A
A 17 B B
A 17 B C
A 18 B A
A 18 B B
A 18 B C
A 19 B A
A 19 B B
A 20 B A
A 20 B B
A 20 B C
A 21 B A
A 21 B B
A 21 B C
B 5 B A
B 5 B B
B 5 B C
```

For group A: I want to test the difference of the difference:

```
(GroupA.WhenB.TimeB-GroupA.WhenB.TimeA) - (GroupA.WhenA.TimeB-GroupA.WhenA.TimeA)
```

For group B: then same thing:

```
(GroupB.WhenB.TimeB-GroupB.WhenB.TimeA) - (GroupB.WhenA.TimeB-GroupB.WhenA.TimeA)
```

Then, I want the difference between the difference of difference:

```
((GroupB.WhenB.TimeB-GroupB.WhenB.TimeA) - (GroupB.WhenA.TimeB-GroupB.WhenA.TimeA)) - ((GroupA.WhenB.TimeB-GroupA.WhenB.TimeA) - (GroupA.WhenA.TimeB-GroupA.WhenA.TimeA))
```

NB! Subject 19 is missing two libraries, one at for WhenA.TimeC and one at WhenB.TimeC.

So, how do I make my design matrix, correcting for paired samples? I have tried this:

```
full.m <- model.matrix(~ Group + Group:ind.n + Group:Time:When, dt.ss)
ss1 <- which(apply(full.m, 2, sum) == 0)
full.m <- full.m[,-ss1]
colnames(full.m)
library(caret)
findLinearCombos(full.m) # Remove linear combinations
full.m <- full.m[,-c(37:38)]
dds <- DESeqDataSetFromMatrix(countData = as.matrix(d.ss),
colData = dt.ss,
design = ~ 1)
dds <- DESeq(dds, full=full.m, parallel=TRUE, betaPrior=FALSE)
```

Questions:

1) How do I specify the wanted results?

2) Why did I have to remove terms 37 and 38? How do I now specify them?

Here an attempt:

```
results(dds, contrast=list("GroupB.timeB.whenB","GroupB.timeB.whenA"))
results(dds, contrast=list("GroupA.timeB.whenB","GroupA.timeB.whenA"))
```

Thus, I think I got the first order interactions. However, how do I get the second order interaction?:

```
results(dds, contrast=list(c("GroupB.timeB.whenB","GroupB.timeB.whenA"), c("GroupA.timeB.whenB","GroupA.timeB.whenA")) ???????
```

**24k**• written 4 months ago by Pindre •

**0**