Crystal,

If you provide a gene annotation to Cufflinks, the transcripts produced will match those in the annotation exactly. If you assemble without a gene annotation, the transcripts produced will match the reference in some cases, but, in others, will not match the reference due to small and/or large errors. Because '=' denotes an exact match between an assembled transcript and a reference transcript, more '=' are to be expected when Cufflinks has a gene annotation.

Finally, a couple procedural issues:

*please send questions about analyses and tool usage to the galaxy- user mailing list, not galaxy-dev or individual developers; *please do not send duplicate emails as it can confuse our tracking system and slow down our response rather than speed it up.

Good luck,
J.

On Aug 17, 2011, at 9:14 AM, Crystal Goh wrote:

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
<Workflow and parameter for 2 approaches.zip><Approach 1 Transcript FPKM tracking (Cufflinks with reference annotation).zip><Approach 2 Transcript FPKM tracking (Cufflinks without reference annotation).zip>

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