25 DE genes with 8 samples and 4 DE genes with 16 samples
0
0
Entering edit mode
6.1 years ago
Pin.Bioinf ▴ 340

Hello, I am doing a differential expression analysis, and first my group sequenced 8 samples of responders and non responders to a treatment, and asked that I analyze and get the DE genes (25 DE genes). After that, they sequenced 8 samples more, and now we get 4 DE genes, no gene matches the DE genes in the results for the previous batch alone.

They ask if this is normal, and I know that the more samples we have, the true DE genes will be reasured and the not really importantly DE genes are dilluted as more samples are added.

Is all this normal?

RNA-Seq DESeq2 • 2.2k views
ADD COMMENT
1
Entering edit mode

Which software are you using for finding DEGs? Maybe it would be helpful to compare different software outputs. How these samples are placed in a PCA plot? I would also look into the read count for individual replicates and see how the read counts are different in each replicate of the 4 DEGs. I use CummeRbund for such analysis (http://compbio.mit.edu/cummeRbund/manual_2_0.html).

ADD REPLY
1
Entering edit mode

Did you add a batch effect in the (second) model?

ADD REPLY
0
Entering edit mode

You mean add the batch parameter to the formula? Is that enough to solve batch effect?

ADD REPLY
1
Entering edit mode

It seems you have two different sequence runs, so a batch effect. In the formula yes with DEseq2. Did you explore your data? With MDS plots? Boxplots? Etc.

ADD REPLY
0
Entering edit mode

Yes, I did. By adding the batch information to the formula I only get 1 DE gene now!

ADD REPLY
0
Entering edit mode

How do the responder and non responder samples cluster in the MDS plot? Please show the plot in your question.

ADD REPLY
0
Entering edit mode
ADD REPLY
0
Entering edit mode
ADD REPLY
0
Entering edit mode

What is your own interpretation of the PCA? Do you expect DE genes between GOOD and BAD samples?

ADD REPLY
0
Entering edit mode

It does not seem like there is a difference as good and bad samples do not appear even close to each other, so I think the reason i dont get any DE genes is because there are none that are significant enough. But as I am new to this and the biologist is saying that there is no way there are no DE genes, I dont know what to do. Maybe it can also be of interest for this matter that the M per read mapped were really bad, like 0.7 M, 1, 6 M per read in many samples.

ADD REPLY
1
Entering edit mode

It is hard to tell what went wrong, I don't know the details (talk with the sequence facility about the quality of your data). It could be technical or real biological (I can't judge from the info you give). But from the PCA I also conclude there is not much difference between GOOD and BAD, so only a handful of (false positive) DE genes seems logical.

ADD REPLY
1
Entering edit mode

Thank you for all your help

ADD REPLY

Login before adding your answer.

Traffic: 1861 users visited in the last hour
Help About
FAQ
Access RSS
API
Stats

Use of this site constitutes acceptance of our User Agreement and Privacy Policy.

Powered by the version 2.3.6