Hello Lizex, The utility tools from Tophat are not included with Galaxy, so that is perhaps why you are having trouble creating junctions files, but I am wondering if you need them at all. You mention that you have different sequence types from different samples. These represent different conditions? Or some do and others are replicates or complete replacements for the originals (the re-sequenced data)? For data representing different samples/conditions, you would not want to map the data together with Tophat or run it together in Cufflinks. Even replicates are not mapped in the same Tophat job, although they are included in the same Cufflinks job. The tool authors have an opinion about the value of replicates - so be sure to read about that at the Cufflinks web site. http://cufflinks.cbcb.umd.edu/howitworks.html#reps The first time different conditions would be in the same job would be at the stage where Cuffmerge is run, to prepare for Cuffdiff - where the differential expression analysis would take place. This is past the stage where individual reads are involved. Our RNA-seq tutorial is here: https://main.g2.bx.psu.edu/u/jeremy/p/galaxy-rna-seq-analysis-exercise And several donated by the Galaxy community are here: http://wiki.galaxyproject.org/Learn#Other_Tutorials But the best resource is the paper from the tool authors ("Protocol"): http://tophat.cbcb.umd.edu/manual.html http://cufflinks.cbcb.umd.edu/manual.html In the end, you may decide that some of the complexity of your data can be reduced by dropping some datasets, to simplify and achieve the same overall results, or perhaps even improved results. In general, I think it is probably safe to say that the less "done" to prep expression data, and certainly the more homogeneous it is, the better the result. But this is your decision. Good luck with your project. Next time when asking a question, please open a brand new email message and start a new thread, not reply to an existing thread and just change the subject line. This helps us with tracking and is appreciated. Thanks! Jen Galaxy team On 2/16/13 5:23 AM, Lizex Husselmann wrote:
Dear all
I have in my project single end reads (50 bp) for some samples and paired end reads (100 bp frw and rev) for the other samples. I had to re-sequence some of the samples of which I have paired-end reads. However the re-sequence data I receives is single-end reads of 250 bp. Tophat wont allow mapping single and paired-end reads together. It says the result will look bad. They do mention that you can convert the junctions.bed file (output of Tophat) with bed_to_juncs using the -j option. I quote for TopHat manual "run TopHat on the 2nd set of reads using the -j option to supply the junctions file produced by be_to_juncs in the previous step". This is supposed to work but I didn't get it to work. Any suggestion on how I should go about analyzing this data?
Kind regards
Lizex
Nate Coraor <nate@bx.psu.edu> 02/15/13 5:48 PM >>> On Feb 15, 2013, at 10:35 AM, Mike Dufault wrote:
To whom it may concern:
The History panel on the right side of the Galaxy page is taking a very very long time to load. Also, when it does load, I have tired to save my .bam files and the transmissions gets truncated to ~7000kb - 8000kb of data. All of my .bam files are several GB.
Some times, when I retry tor download the data, it succeeds and other times it is again truncated. The size of the truncation may be different for the same file on the retry attempt.
Is there a problem with Galaxy?
Hi Mike,
There are some performance problems with the Main site that we are currently investigating Thanks for the information and we apologize for the problems.
--nate
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___________________________________________________________ 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:
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