Hi Ateequr, I don't think there is an easy answer to your question. I would highly recommend you take a look to this recently released paper from Tophat and Cufflinks authors: Differential gene and transcript expression analysis of RNA-seq experiments with TopHat and Cufflinks. http://www.ncbi.nlm.nih.gov/pubmed/22383036 The somehow short answer to your question is, you should run cuffdiff with the transcript file from cuffmerge, although I don't quite understand what you mean by "and concatenate data sets". You should not concatenate the BAM files, or you won't be able to run cuffdiff correctly. Depending on how you ran cufflinks, by using the transcript file from cuffmerge you will get expression values for possible new transcripts not present in the databases. This is commonly desired. If you aren't interested in possible new transcripts, then you could simply use a GFF file with transcripts definitions for the genome you are using. In this case you will have the advantage of getting the results with IDs compatibles with the database source the GFF came from. If you use the cuffmerge file you might have the results using cufflinks generated IDs. Hope it helps, Carlos On Tue, Apr 17, 2012 at 11:00 AM, Ateequr Rehman <ateeqrr@yahoo.com> wrote:

Dear All I have simple and question for cuffdiff should we run cuffdif on merge transcript file (produced by cuffmerge) and concatenate data sets or directly on cufflink produced files, in the later case, i have two transcript files resulting from cufflink on sample 1 and 2 respectively, result using sample 1 as transcripts are not the same when i am suing sample 2 as transcript

i am bit confused what should be the correct way

any help is very much welcomed

Best ateeq

Ateequr Rehman House No. 2 ground floor Blauenstr. 10 79115 Freiburg im Breisgau

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