Re: [galaxy-user] How should I include biological replicates in cufflink/cuffdiff?
I am dealing with a bacterium which has about 4000 genes. When I tried Cuffmerge to merge everything with reference annotation, I got a merged file of only 50 lines. If I left out the reference annotation file, Cuffmerge returned me a merged file of 4000 lines (which is more reasonable). However this difference didn't happen if I use Cuffcompare to merge all the files. With or Without reference annotation, the merged file are both of 4000 lines. If I continue to Cuffdiff with this Cuffcompare file, I got over 1000 significantly changed genes. Could you give me some suggestion on this? Should I just trust the Cuffcompare file? Cuffmerge attempts to remove incomplete or spurious transcripts. My best guess is bacterial transcripts, with few/no introns, are being filtered out because they appear to be incomplete to Cuffmerge. So, in your case, Cuffcompare could be the superior option. You might want to verify my guess by discussing the issue with the cufflinks developers directly: tophat.cuffli...@gmail.com ; please feel free to post anything you learn to this list. Best, J. ___ The Galaxy User list should be used for the discussion of Galaxy analysis and other features on the public server at usegalaxy.org. Please keep all replies on the list by using reply all in your mail client. For discussion of local Galaxy instances and the Galaxy source code, please use the Galaxy Development list: http://lists.bx.psu.edu/listinfo/galaxy-dev To manage your subscriptions to this and other Galaxy lists, please use the interface at: http://lists.bx.psu.edu/
Re: [galaxy-user] How should I include biological replicates in cufflink/cuffdiff?
Dear Jeremy, Thank you for your advice! However when I tried this out I got some more questions. I am dealing with a bacterium which has about 4000 genes. When I tried Cuffmerge to merge everything with reference annotation, I got a merged file of only 50 lines. If I left out the reference annotation file, Cuffmerge returned me a merged file of 4000 lines (which is more reasonable). However this difference didn't happen if I use Cuffcompare to merge all the files. With or Without reference annotation, the merged file are both of 4000 lines. If I continue to Cuffdiff with this Cuffcompare file, I got over 1000 significantly changed genes. Could you give me some suggestion on this? Should I just trust the Cuffcompare file? Is it possible that there might be some problem with my reference annotation file? Thank you very much, Qian On Sun, Mar 3, 2013 at 9:59 AM, Jeremy Goecks jeremy.goe...@emory.eduwrote: My question is, if I need to compare between 5 time points, should I do comparison pairwise? No, do them all at once with Cuffdiff: (a) set 'Perform Replicate Analysis' to 'Yes'; (b) create 5 replicate conditions, one for each time point; (c) add your replicates for each time point. There's a Cuffdiff flag to do time series analysis, but it isn't implemented yet in Galaxy, so you'll get pairwise comparisons for all conditions. You can use the filtering tool to reduce Cuffdiff outputs to only the timepoint comparisons. I will use cuffmerge to merge 0hour-1, 0hour-2, 0hour-3, 1hour-1,1hour-2.1hour-3 to generate one cuffmerge file. Correct. Then I will run cuffdiff using the merged file, include two groups, group 1 is 0 hour (add 0hour 1-3 in group 1) and group 2 is 1hour (add 1hour1-3 in group 2). Use the process I described above to do all pairwise comparisons in one run. Good luck, J. -- Qian Dong Bauer Lab, MCBD Simon Hall: 313-317 212 S. Hawthorne Dr. Bloomington, IN 47405 Email:do...@indiana.edu Lab Phone:812-855-8443 ___ The Galaxy User list should be used for the discussion of Galaxy analysis and other features on the public server at usegalaxy.org. Please keep all replies on the list by using reply all in your mail client. For discussion of local Galaxy instances and the Galaxy source code, please use the Galaxy Development list: http://lists.bx.psu.edu/listinfo/galaxy-dev To manage your subscriptions to this and other Galaxy lists, please use the interface at: http://lists.bx.psu.edu/
Re: [galaxy-user] How should I include biological replicates in cufflink/cuffdiff?
My question is, if I need to compare between 5 time points, should I do comparison pairwise? No, do them all at once with Cuffdiff: (a) set 'Perform Replicate Analysis' to 'Yes'; (b) create 5 replicate conditions, one for each time point; (c) add your replicates for each time point. There's a Cuffdiff flag to do time series analysis, but it isn't implemented yet in Galaxy, so you'll get pairwise comparisons for all conditions. You can use the filtering tool to reduce Cuffdiff outputs to only the timepoint comparisons. I will use cuffmerge to merge 0hour-1, 0hour-2, 0hour-3, 1hour-1,1hour-2.1hour-3 to generate one cuffmerge file. Correct. Then I will run cuffdiff using the merged file, include two groups, group 1 is 0 hour (add 0hour 1-3 in group 1) and group 2 is 1hour (add 1hour1-3 in group 2). Use the process I described above to do all pairwise comparisons in one run. Good luck, J. ___ The Galaxy User list should be used for the discussion of Galaxy analysis and other features on the public server at usegalaxy.org. Please keep all replies on the list by using reply all in your mail client. For discussion of local Galaxy instances and the Galaxy source code, please use the Galaxy Development list: http://lists.bx.psu.edu/listinfo/galaxy-dev To manage your subscriptions to this and other Galaxy lists, please use the interface at: http://lists.bx.psu.edu/
