Hi,
We have a local install of galaxy and I'm trying to add the reference index 
files for bwa using the information provided in the following link
http://wiki.g2.bx.psu.edu/Admin/NGS%20Local%20Setup

I have modified the bwa_index.loc file present in the ../tool-data directory by 
adding the path to where the index is on our server (Also attached). However, 
even after restarting the server, the reference genome does not show when 
choosing the "use a built-in index option". I'm not sure whether the loc file 
is correctly created and whether any other configuration file needs to be 
changed/updated. Help in the matter greatly appreciated.

Thanks,
Aarti

From: galaxy-user-boun...@lists.bx.psu.edu 
[mailto:galaxy-user-boun...@lists.bx.psu.edu] On Behalf Of Jennifer Jackson
Sent: Thursday, July 05, 2012 1:23 AM
To: Lindsey Kelly
Cc: galaxy-user@lists.bx.psu.edu
Subject: Re: [galaxy-user] Initial QC and grooming for Illumina HiSeq2000 
paired end RNAseq data

Hello Lindsey,

Yes, you have this correct. The general path would be to:

 - join forward and reverse data per run
 - run FASTQ Groomer & FastQC
   (note: if your data is already in Sanger FASTQ format with Phred+33 quality 
scaled
   values, the datatype '.fastqsanger' can be directly assigned and the FASTQ 
Groomer
  step skipped. This is likely true if your data is a from the latest CASAVA 
pipeline, but
   please double check.)
 - discard data as needed based on quality
 - split forward and reverse data that passes QC
 - concatenate all forward reads from a sample into one FASTQ file
 - concatenate all reverse reads from a sample into one FASTQ file.
 - for each sample, run TopHat using the two concatenated FASTQ files

To manipulate paired end data, please see the tools -> NGS: QC and 
manipulation: FASTQ splitter & FASTQ joiner.

To combined data files head-to-tail from multiple runs into a single FASTQ file 
please see the tool -> Text Manipulation: Concatenate datasets.

I am not sure of the actual volume of data, but if these start to get large or 
TopHat errors with a memory problem, a local or cluster instance would be the 
recommendation: http://getgalaxy.org

For reference:
http://tophat.cbcb.umd.edu/manual.html
http://www.nature.com/nprot/journal/v7/n3/full/nprot.2012.016.html

Hopefully this helps. Others are welcome to post comments/suggestions.

Jen
Galaxy team
On 7/2/12 11:17 AM, Lindsey Kelly wrote:
I am trying to do RNAseq analysis on Paired end data from the Hiseq2000.  I 
have about 50 files for each sample (25 forward and 25 reverse - although each 
sample has a different number of files).
I think that I need to:
-convert them into FASTQ sanger format using the FASTSQ groomer tool
-check the quality using the FASTQqc tool

I don't know how to handle this many files.  Do I have to groom and run the QC 
for each file? Should I join the paired files and run both tools on each pair, 
or should I combine all of the data for each sample (which I don't know how to 
do) and then groom and run the QC for all of the reads for the sample.

Thanks in advance for advice
Lindsey




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