This looks like:
Sample Condition
Condition1_rep1 ConditionA
Condition1_rep2 ConditionA
Condition1_rep3 ConditionA
Condition1_rep4 ConditionA
Condition1_rep5 ConditionA
Condition2_rep1 ConditionB
Condition2_rep2 ConditionB
Condition2_rep3 ConditionB
Condition2_rep4 ConditionB
Condition2_rep5 ConditionB
If so, the design is ~ Condition
and the above table would be what DESeq2
accepts as colData
. This is sufficient to indicate the samples that belong to one group. Group membership is not indicated via the design formula as you did with pairs
. If you want to include batch
then add this as an additional column and use ~ batch + condition
. Check by PCA first if you see signs of batch effect. If not leave it out. I hope you did not extract all of ConditionA on day1 and B on days?