Dear all,

I've a dataset of around 150 RNAseq from different patients and after performing quantification of transcript with Salmon. I'm interested in founding immunologic signature correlate with the expression of a specific gene isoform.

So I've retrieve the abundance (TPM) of that isoform among my 150 RNAseq and I'm using this as a continuous phenotype into GSEA.

Then I've performed gene summarization of my Salmon output using tximport and I'm using this as my expression table (TPM) for GSEA.

Since I've only one replicate for each phenotype I'm trying to found the best method to rank my genes. I've tried to performed a DESeq2 analysis of my dataset using again the abundance of that isoform as my condition and taking the wald score to rank my genes.

Overall I'm not confident I'm taking the best approach to found those signature correlate to the expression of that specific isoform and seeking for your help.

Best Regards,

Leo

My approach would probably be to compute the vst() of the dataset and then rank transcripts/genes by correlation to the transcript of interests.