Hi Jen, Thank you for the information regarding the FastQ information. It was really helpful. Lately, I have been getting the following error: "Error getting history update from this server- Bad Gateway". This occurred after I tried to reupload some pre-aligned/ and indexed BAM files from NCBI GEO because I was hoping to generate and retrieve FPKM/RPKM values from them. Unfortunately, the my old files are still not available on Galaxy and I get an Internal Server Error when trying to retrieve them. Although I can get the work flow for them. The last weird error is that when I use Cuffdiff, I get FPKM of 0 with p/q values of 1 all the time. When this should not be the case as the BAM files are from two different organs. This is for every single gene, hence this indicates that something is wrong. I was able to retrieve the GTF file from UCSC main with the following settings: Insect - D. pseuddobscura Group - Genes and Gene Prediction Tracks Track: Flybase Table FlybaseGene Output format: GTF. I was wondering should these setting be fine or should I change the Group to mRNA or some other settings. Although the one that is avilable on UCSC is old dp3 file from 2004. The latest GFF is 3.1 on Flybase. I was wondering anyway to convert to a GTF file. Sorry for so many questions. Thank you again for the great help. Sincerely, Zain ________________________________ From: Jennifer Jackson [jen@bx.psu.edu] Sent: Tuesday, May 07, 2013 3:21 PM To: Zain A Alvi Cc: galaxy-dev@bx.psu.edu Subject: Re: [galaxy-dev] Tophat problem Hi Zain, I believe we already worked out the .fastqsanger/grooming part of this question in another thread. But for others reading this post, this is a help link: See "FASTQ" http://wiki.galaxyproject.org/Support#Dataset_special_cases Our RNA-exercise covers and example workflow: https://main.g2.bx.psu.edu/u/jeremy/p/galaxy-rna-seq-analysis-exercise Best, Jen Galaxy team On 5/3/13 8:59 PM, Zain A Alvi wrote: Dear Sir or Madam, I hope this reaches you well. Lately, I have been trying to use tophat and then use bowtie on Galaxy project to create an aligned BAM file. The original data came from a SRA file that I have acquired from the Japanese DNA Databank. This SRA was then converted to FASTQ using the tools available on Galaxy project. Now when I go under Tophat on Galaxy Project, I am unable to select the converted RNA-Seq FASTQ file. I was wondering, is there a specific format for the file to be in. Currently it is just a *.fastq file. I am confused as to why I am not being able to select the FASTQ file. Also if there is a guide on how to use Galaxy Project to create an aligned BAM file and then check for expression through Cufflinks package. I would really appreciate it. Sincerely, Zain ___________________________________________________________ Please keep all replies on the list by using "reply all" in your mail client. To manage your subscriptions to this and other Galaxy lists, please use the interface at: http://lists.bx.psu.edu/ To search Galaxy mailing lists use the unified search at: http://galaxyproject.org/search/mailinglists/ -- Jennifer Hillman-Jackson Galaxy Support and Training http://galaxyproject.org