Hi, I am Crystal. I have some problem with Cuffdiff output. Hope can get some advice. Thanks.
After aligning RNA-seq reads with Tophat, I used the Tophat output for Cufflinks.
For Cufflinks, I tried two approaches and compared the results: 1st approach: Put zebrafish Ensembl GTF as reference annotation 2nd approach: without reference annotation.
From the output of above 2 approaches, I continued with Cuffcompare (with reference annotation) and Cuffdiff,
Attached word document is the workflow and parameters I set for these 2 approaches.
When I compared the output of Cuffdiff between these 2 approaches, a total of 48584 tracking id with class code "=" was observed in trancript FPKM tracking file from Approach 1, whereas there is only 1248 tracking id with class code '=' from Approach 2 (I attached transcript FPKM tracking files from approach 1 and 2)
In my opinion, I should observe 48584 tracking id with class code '=' and additional tracking id with other class codes in transcript FPKM tracking file from Approach 2.
Can I get advice on this?
Thank you.
Best regards, Crystal