I am trying to use Cuffdiff to find significant changes in mRNA transcript expression between two conditions (1. silenced gene, 2. overexpressed gene) but for some reason have been having difficulties using the Cuffmerge output gtf as input into Cuffdiff. This seems to be a basic question with most likely an easy answer, but I was hoping someone could help me as I have not been able to figure it out on my own and have been trying different things with no success.
This is what I have done so far:
1. Mapped the reads for the two samples individually using Tophat.
2. Ran Cufflinks on each dataset produced by Tophat using a reference annotation downloaded from UCSC to assemble the reads.
3. Ran Cuffmerge by uploading the two GTF "assembled transcript" files from the two samples. The one produced output file consisted of merged data from the two cufflink runs.
4. Attempted to run Cuffdiff by uploading the Cuffmerge GTF output and assigning Conditions #'s 1 and 2 their respective Tophat assembly files. Cuffdiff could not run.
Instead I have uploaded the reference annotation I downloaded from UCSC (in place of the Cuffmerge output file) to Cuffdiff and assigned Conditions #'s 1 and 2 the same file produced by Tophat on condition #1 (silenced). A tabular file was produced that contained the same quantities for values 1 and 2 for the two conditions per gene as expected. This meant that the log2(fold_change) = 0 and there was no significant difference in the t-test results.
I then uploaded the same reference annotation from UCSC and assigned Conditions #'s 1 and 2 the same file produced by Tophat on condition #2 (over-expressed). A tabular file was produced. Again, the quantities for values 1 and 2 were the same per gene.
Since I could not get both condition files uploaded to Cuffdiff, I was planning to look at the two different tabular output files and do my own comparison using EXCEL. But I would prefer to run Cuffdiff's t-test significant difference test and follow the simple flow Tophat --> Cufflinks --> Cuffmerge --> Cuffdiff?
Can someone identify what I did wrong? Please let me know if I should clarify any of my steps.
Thank you so much. I really appreciate the help.