Dear the bioinformaticians,

I am using DESeq2 to normalize data from 100 samples from 100 patients. The 100 samples are classified into 3 groups (group A; 30 samples, group. B: 30 samples, group C: 40 samples). I want to compare significance of gene expression among each group. In statistics, we use ANOVA, but in DESeq2 there is no option to compare gene expression among more than three groups.

- So, my first question is "Is it ok to 1) obtain the normalization scale factors using 100 samples, 2) multiply normalisation scale factors to each one, and 3) compare the gene exrpression among three groups.". If not, is there any suggested way to compare multi group analysis?

- To compare group A and group B, is it OK to use the normalised counts using all 100 samples? Or should I run DESeq2 with only group A and group B (30+30 = 60 samples). Regarding the concept of DESeq2, both methods (calculating scale factors from 100 samples and 60 samples) would not show big difference.

Thank you for your comments and advice.

```
# include your problematic code here with any corresponding output
# please also include the results of running the following in an R session
sessionInfo( )
```

I appreciate your kind reply. All my questions were solved. Thank you very much again.