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,

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

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