Hi,

I need help the analysis of my RNA-seq samples using DESeq2. This is the first time I am doing this kind of analysis. So, I need some help with my data. I have gotten some results but I`m not sure if it`s correct.

This is my experimental design:

coldata
#                       group      treatment ID
#Ctr_untreated_1-1      siCtr      untreated  A
#Ctr_untreated_1-2      siCtr      untreated  A
#Ctr_untreated_2-1      siCtr      untreated  B
#Ctr_untreated_2-2      siCtr      untreated  B
#Ctr_untreated_3-1      siCtr      untreated  C
#Ctr_untreated_3-2      siCtr      untreated  C
#Ctr_untreated_4-1      siCtr      untreated  D
#Ctr_untreated_4-2      siCtr      untreated  D
#KD_untreated_1-1       siX        untreated  E
#KD_untreated_1-2       siX        untreated  E
#KD_untreated_2-1       siX        untreated  F
#KD_untreated_2-2       siX        untreated  F
#KD_untreated_3-1       siX        untreated  G
#KD_untreated_3-2       siX        untreated  G
#KD_untreated_4-1       siX        untreated  H
#KD_untreated_4-2       siX        untreated  H
#Ctr_treated_1-1        siCtr hormonetreated  I
#Ctr_treated_1-2        siCtr hormonetreated  I
#Ctr_treated_2-1        siCtr hormonetreated  J
#Ctr_treated_2-2        siCtr hormonetreated  J
#Ctr_treated_3-1        siCtr hormonetreated  K
#Ctr_treated_3-2        siCtr hormonetreated  K
#Ctr_treated_4-1        siCtr hormonetreated  L
#Ctr_treated_4-2        siCtr hormonetreated  L
#KD_treated_1-1         siX   hormonetreated  N
#KD_treated_1-2         siX   hormonetreated  N
#KD_treated_2-1         siX   hormonetreated  O
#KD_treated_2-2         siX   hormonetreated  O
#KD_treated_3-1         siX   hormonetreated  P
#KD_treated_3-2         siX   hormonetreated  P
#KD_treated_4-1         siX   hormonetreated  Q
#KD_treated_4-2         siX   hormonetreated  Q

I have 2 questions regarding the analysis with DESeq2.

1: I would like to find differentially expressed genes in KD_treated compared with Ctr_treated. What should the script look like if I keep all samples (including Ctr_untreated and KD_untreated)?

2:  A second scenario is to find differentially expressed genes in KD_treated compared with all other samples?

Thanks in advance

Zeynep

So after you collapse the technical replicates, you will have half the columns of dds I am assuming.

To make the comparisons (1) and (2) you can follow this advice about constructing a "group" variable, which combines KD vs control and hormone treated vs untreated information.

http://bioconductor.org/packages/release/bioc/vignettes/DESeq2/inst/doc/DESeq2.html#interactions

Thank you very much for the feedback!