I'm dealing with alternative splicing and isoform switching. I am trying to find interesting candidates for my research from a transcriptome analysis. We run Cufflinks/Cuffdiff2 to estimate differential expression of genes, isoforms, cds (coding sequence).
For what I understand in an alternative splicing perspective the most interesting aspect to consider is not the differential expression per se but the switch in expression, i.e. how much the proportion among different cds and isoform expression of a gene change between 2 conditions. Cuffdiff automatically calculate the significance in this switch, giving cds.diff, splicing.diff (for isoforms sharing the same transcription start site) and promoter.diff (for isoforms which uses different transcription start site). I'm focusing on genes which show a different proportion in the cds expression, so I'm analyzing the genes from the cds.diff., regardless of the specific event which causes the difference in cds.
Now I want to know which specific cds are mainly responsible for the change in cds proportion, for example which cds significantly augment its proportion in condition 2 as compared to condition 1. I tried to select those cds which show a significant increase in expression (data from the cds.exp.diff) and a log2FC above a determined threshold, but this yield also cds which increase in expression but decrease in proportion (for example a cds double it's expression but it goes from being the 70% to being the 50% of the total cds expressed by the gene in the 2 conditions).
So I'm thinking that the most relevant information would be the change in proportion, but I don't know how to define it...would it be a 10% different significant? how to correct this parameter for the actual expression? I mean a 10% difference in an low abundant cds/isoform would not be as relevant as a 10% in an high expressed one (and moreover the expression is estimated in FPKM by Cuffdiff). And how should I consider those cds which don't change expression per se but change the proportion, as a result of slightly changes in expression of the other cds of the gene (I found some genes which are present in the cds.diff files, so which undergo a significant switch, but for which none of the cds are detected as significantly differentially expressed)?
note. of course I subset the genes in which the parameter significant is "yes" in the cds.diff file
Thanks in advance!