Search
Question: help DESeq2 model design
2
gravatar for euge.eugenio
5 months ago by
euge.eugenio20
euge.eugenio20 wrote:

Hi, I'm new to bioinformatics so sorry if the question is a bit naive.

i've been trying to understand how to design a model matrix for deseq2 in this experiment design

control 2h
control 2h
control 2h
control 2h
control 2h
control 2h
control 6h
control 6h
control 6h
control 6h
control 6h
untreated 0h
untreated 0h
untreated 0h
untreated 0h
untreated 0h
untreated 0h
wnt3a 2h
wnt3a 2h
wnt3a 2h
wnt3a 2h
wnt3a 2h
wnt3a 2h
wnt3a 6h
wnt3a 6h
wnt3a 6h
wnt3a 6h
wnt3a 6h
wnt3a 6h

 

basically the untreated is different than control because control is still treated with media without wnt3a

I'm having difficulties representing this data in a correct design formula for deseq2 because obviously the condition untreated is linearly dependend to timepoint 0. i can use ~timepoint + condition if i set the untreated to control but i think the correct formula for this experiment would be something like ~timepoint + condition + timepoint:condition, that as i understand means that the effect of the timpoint depends on what condition it is. the problem is that now the matrix is not full rank again. splitting the controls between the two conditions doesn't really seem right to me as i would have 3 replicates each instead of 6.

any suggestions? 

thanks

 

ADD COMMENTlink modified 5 months ago by Michael Love19k • written 5 months ago by euge.eugenio20
1
gravatar for Michael Love
5 months ago by
Michael Love19k
United States
Michael Love19k wrote:

“the correct formula for this experiment”: this depends on what you are looking to test. Can you say what you are looking to test here? Eg “we want to find genes such that...”

ADD COMMENTlink written 5 months ago by Michael Love19k

This is just some test data i downloaded from a public repository, but my idea would be to see for example which genes get activated during wnt3a activation over time, so differentially expressed genes from time 0 to time point 2, and from time point 0 to 6 compared to control. I also wanted to see if i could apply the time course part on the RNA-seq workflow tutorial. so time point 0, 2 and 6 separating the two conditions.

Thanks again

ADD REPLYlink written 5 months ago by euge.eugenio20

Here's a tricky thing, which catches lots of folks: let's say we have group = control, A, B, and C. What does it mean to say B vs A compared to control? Usually people want (B-control) - (A-control). This ends up being equivalent to B - A. It would be different if you had (B-control_B) - (A-control_A), and we have different ways of doing this design, but with a single control, it drops out of the equation, so you really just need to add a new variable 'group' where you combine the condition and time point above, and then use ~group as a design.

ADD REPLYlink modified 5 months ago • written 5 months ago by Michael Love19k

ok so if i understood correctly i should just combine the two columns together and call it as a single variable and then run DESeq with just one variable right?

ADD REPLYlink written 5 months ago by euge.eugenio20

Yes, that's easiest here.

ADD REPLYlink written 5 months ago by Michael Love19k

Perfect, Thanks!

ADD REPLYlink written 5 months ago by euge.eugenio20
Please log in to add an answer.

Help
Access

Use of this site constitutes acceptance of our User Agreement and Privacy Policy.
Powered by Biostar version 2.2.0
Traffic: 368 users visited in the last hour