what to do when adjusted p-values are not acceptable in RNASeq?

Question

[nazaninhoseinkhan]

Dear all,

I have RNASeq data of 2 normal and 5 cancer tissues.

Although the quality of reads were very good, when I run Deseq2 the adjusted p-values was not acceptable at all (only 3 genes with adjusted p- value less than 0.05).

Can you guide me how can I select differentially expressed genes in this situation?

Regards

Nazanin

[JC]

Cancer is really diverse in gene expression, could be better to analyze your cancer samples individually using the normals as "control", also you probably want to try another package as EdgeR to see if this is something particular in your dataset.

[Panagiotis Moulos]

You can also try an integrated approach like metaseqR with more than one algorithms and combined p-values which may partially remedy your problem. Disclaimer: I am the author of that package.

[Asaf]

Take a look at the correlations between the expression levels of the cancer samples (and control). You can then exclude samples that look very different from the rest or split them into two groups (according to their similarity or type).

[Matteo Schiavinato]

How are you counting the reads that map on each feature? HTSeq? If yes, how many reads you have in the "NoFeature"? Are they a large fraction of your dataset?

Answer 1

[nazaninhoseinkhan]

Dear JC, Panagiotis, Asaf and Macspider,

Thank you so much for your comments.

I re-run the Deseq2 after calculating read numbers using Htseq Count and the result was OK this time.

I realized that the first time I had introduced the Normal and Cancer groups wrongly.

I hope everything goes well with you

Nazanin