I have couple of questions regarding cufflinks. I have done cufflinks transcripts assembly for 12 cell lines from human.And merged all the transcript.gtf files from all samples using the reference transcript file, by cuffmerge and then did cuffdiff analysis. This analysis gave 163 genes deferentially expressed from around 60k total list of genes. The output files in the cuffdiff analysis does not give per sample read count and fpkm information so I was not sure how to validate the the significant genes by comparing the read counts in each sample. Also, the publication by trapnell group, does mention that for a well annotated organism like human , mouse etc, its not important to carry out the transcript assembly if looking at the differential expression, so I followed the Alternation protocol mentioned in that paper where the alignment is done by turning off the novel splice junction detection in tophat and then running cuffdiff analysis by skipping the transcript assembly. This analysis gave 141 genes diferentially expressed but again I don't know how to get the per sample read count and fpkm data from cufflinks pipeline. The output files of cuffdiff from my first type of analysis (transcript assembly) does not give any significant differential spliced genes. Is this normal?(I did reference guided assembly and tophat was run with novel splice junctions option on).
The cufflinks pipeline, although mentioned to be robust in many papers, does not seem to be transparent in what its doing. I am wondering if I should choose some other method of differential expression analysis than cufflinks. Any suggestions on this will be helpful.