**0**wrote:

Hello,

I am trying to perform DE gene analysis on RNA-Seq data. My groups consist of animals from two different populations (SD v. XE) and two different treatments (CON v. E2). I'm following section 3.3.1 in the edgeR user guide.

My metadata is as follows:

` samples treat pop group`

1 THXL1A E2 SD E2.SD

2 THXL1B CON SD CON.SD

3 THXL1C CON XE CON.XE

4 THXL1D E2 XE E2.XE

5 THXL1E CON SD CON.SD

6 THXL1F E2 SD E2.SD

7 THXL7 CON XE CON.XE

8 THXL8 E2 XE E2.XE

9 THXL9 CON SD CON.SD

10 THXL10 CON SD CON.SD

11 THXL11 E2 SD E2.SD

12 THXL12 E2 SD E2.SD

I am trying to make the following contrasts:

`colnames(design) <- levels(group) `

`fit <- glmFit(dispData, design)`

`contrasts <- makeContrasts( `

` XE.E2vsCON = XE.E2-XE.CON,`

` SD.E2vsCON = SD.E2-SD.CON,`

` SDvsXE.CON = SD.CON-XE.CON,`

` `

** SDvsXE.E2 = (SD.E2-SD.CON)-(XE.E2-XE.CON)**,

`levels=design)`

I'm having trouble interpreting this specific contrast:

`lrt_treat <- glmLRT(fit, contrast=contrasts[,"`

**SDvsXE.E2**"])

From following the guide, it says that I should be finding genes that have responded differently to the SD and XE with E2 treatment. Does this mean I'm comparing

A) SD-E2 v. XE-E2 (baseline)

or am I looking at

B) SD-E2 and XE-E2 combined v. SD-CON and XE-CON combined (baseline)?

Thanks!

Susan

**39k**• written 3.1 years ago by xluong •

**0**