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 [j...@bx.psu.edu]
Sent: Tuesday, May 07, 2013 3:21 PM
To: Zain A Alvi
Cc: galaxy-...@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



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Jennifer Hillman-Jackson
Galaxy Support and Training
http://galaxyproject.org
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