Question: Determining Replicates For Deseq?
gravatar for Deena
6.9 years ago by
Deena260 wrote:

Hello, I am relatively new to RNA-Seq data analysis so I apologize in advance if this is a novice question. I have been reading several forums on the subject and I think I have the general idea, but I would be happy to get advice from the experts.

I have RNA-Seq data for multiple samples of a specific cancer. Previously from Gene Expression analysis (on Illumina platforms) the lab ran before I arrived, we have learned in that this cancer can be divided into 3 subgroups (1,2,3). I would like to find the list of differentially expressed genes between RNA-Seq samples pertaining to subgroups 2 and 3, using DESeq.

I am somewhat confused here as to what I should consider as my 'biological replicates', or whether I should not consider replicates at all for my analysis.

For each of the subgroups, I only have one sample per patient. So the scenario looks like this:

Subgroup 2: Sample A, Sample B, Sample C, Sample D

Subgroup 3: Sample X, Sample Y, Sample Z, Sample F, Sample W

In this case, should I consider that Samples A,B,C,D are all biological replicates of Subgroup 2, and Samples X,Y,Z,F,W as biological replicates of Subgroup3? Each of the samples in the subgroup pertains to a different patient, and there is no control sample for each patient (and in this case, no pairing between my samples).

If I am to consider this scenario, any advice on the DESeq parameters? Right now I am just running the defaults as appears in the vignette.

The alternative is to consider that I don't have any replicates and run the two groups. So the DESeq for calculating dispersions would be like this:

cds = estimateDispersions( cds, method="blind", sharingMode="fit-only", fitType="local" )

I have tried both scenarios. In the case where I don't consider any replicates at all, I have ended up with a much larger number of differentially expressed genes at p<0.1 (1380 as opposed to 92).

Any advice would be appreciated! Thank you in advance! Deena

ADD COMMENTlink modified 6.9 years ago • written 6.9 years ago by Deena260
gravatar for Deena
6.9 years ago by
Deena260 wrote:

I have also posted this in Seqanswers and received an answer, being followed-up on:

Hopefully it may be useful to others.

ADD COMMENTlink written 6.9 years ago by Deena260

Interesting never even knew that there was a package called DESeq2

ADD REPLYlink written 6.9 years ago by Sudeep1.6k
Please log in to add an answer.


Use of this site constitutes acceptance of our User Agreement and Privacy Policy.
Powered by Biostar version 2.3.0
Traffic: 1571 users visited in the last hour