Hello All,

For my analysis I have three time points Time1, Time2 and Time3 these were measured based upon three different treatments. I am trying to run differential analysis between T1 vs T2, T1 vs T3 and T2 vs T3, I have some technical replicates that I summed using the collapseReplicates() function, the "runsCollapsed" column below describes that.

For running the comparison I have tried using "~SampleName+Status" as my design matrix. This doesn't seem to work and I have the following error:

```
Object <- DESeqDataSetFromMatrix(countData = CountFrame, colData = Pheno , design =
~SampleName + Status)
factor levels were dropped which had no samples
Error in checkFullRank(modelMatrix) :
the model matrix is not full rank, so the model cannot be fit as specified.
One or more variables or interaction terms in the design formula are linear
combinations of the others and must be removed.
Pheno file:
Sample.ID SampleName Well Status runsCollapsed
S11 MS1A C4 Time1 C4,D4
S13 MS1B C5 Time2 C5,D5
S1 MS2A A1 Time1 A1,B1
S3 MS2B A2 Time2 A2,B2
S21 MS5A C9 Time1 C9,D9
S23 MS5B C10 Time2 C10,D10
S25 MS5C C11 Time3 C11,D11
S33 MS7A E4 Time1 E4,F4
S35 MS7B E5 Time2 E5,F5
S37 MS7C E6 Time3 E6,F6
S39 MS8A E7 Time1 E7,F7
S41 MS8B E8 Time2 E8,F8
S43 MS8C E9 Time3 E9,F9
S45 MS9A E10 Time1 E10,F10
S47 MS9B E11 Time2 E11,F11
S49 MS9C E12 Time3 E12,F12
```

Can someone please suggest if there is a better way to do this? Also for Time3 I have lesser samples compared to Time1 and Time2. Could this be the reason for the error?

I think you need to drop "Samplename" from your design, or edit the sample names so they don't have the letters at the end. You don't really have two dimensions of conditions here, because every sample name has one and only one status. If all your samples really are that different, and you don't want to combine them together, you have no replicates, which really limits what you can learn from this experiment.