RNASEQ differential expression of paired samples

Question

[hans]

Hello I have the following experimental design of RNASEQ experiment: 12 samples, 6 are males 6 are females, and two conditions of treatment (Before and After) according to this table:

ID(subject)    sex    treat
1              M      B
1              M      A
2              M      B
2              M      A
3              M      B
3              M      A
4              F      B
4              F      A
5              F      B
5              F      A
6              F      B
6              F      A

I found that there is a large gene expression variation among subjects from the same sex (ID). My assumption, based on biological observations, is that there should be genes that are deferentially expressed according to the treatment in the males but are not deferentially expressed in the females. What would be the right software and model design to find those DEG.

I tried DESeq2 but it did not accept the model design ~ID+sex+treat+treat:sex because the ID is limited to one sex so it is not a full model.

Thank you

Answer 1

[Carlo Yague]

Hi, If I understand correctly, this is a case of nested design. The same ID levels are always either male or female.

> colData
DataFrame with 12 rows and 3 columns
            treat      sex       ID
         <factor> <factor> <factor>
sample1         A        M        1
sample2         B        M        1
sample3         A        M        2
sample4         B        M        2
sample5         A        M        3
...           ...      ...      ...
sample8         B        F        4
sample9         A        F        5
sample10        B        F        5
sample11        A        F        6
sample12        B        F        6

For this specific case, there is a workaround explained in this tutorial: you will need to add a grouping variable in the design.

[hans]

Thank you, The tutorial seems to provide the right solution