Question: Next-Seq 500 data
gravatar for beckylazensky
3.1 years ago by
United States
beckylazensky0 wrote:

Hi! I am new to Galaxy and just received a RNA-Seq dataset generated by an Illumina 'Next-Seq 500'. My files are in SAM format. My goal is to run Cuff diff and look at differential expression patterns between cases and controls. Our lab already ran a few data cleaning and alignment steps before sending me the clean SAM files. Bowtie 2 was run. I still want to learn how to use Galaxy with these data so I can learn some additional tools beyond what they did with the data already. I am attempting to run tophap and cufflinks in galaxy and got an error for both. I did run the groomer and fastqc and these both worked fine on my files. I used groomer to sort by chromosome and it is now in sanger format ACS II range 35-70. When I ran tophap 2, I got an error that said 'tool execution failed'. It also said 'index error; list index out of range'. I am wondering- do I need to still run this since Bowtie 2 was already ran on my data outside galaxy using the command line? Also are the next-seq 500 files files compatible with galaxy?  I tried running cuff link and cuff diff directly without the files and got an error message that says 'cuff link: no update check' and 'cuff diff: command not found'. I appreciate everyone help.


rna-seq • 998 views
ADD COMMENTlink modified 3.1 years ago by Dannon Baker3.7k • written 3.1 years ago by beckylazensky0
gravatar for Dannon Baker
3.1 years ago by
Dannon Baker3.7k
United States
Dannon Baker3.7k wrote:

Is this regarding the Galaxy server at, or has your lab installed a Galaxy there that you're using?

If you're using there were a few galaxy service issues over the holiday break that may have contributed to this, depending on when you had this error.  Can you click the little bug icon on the error'ed dataset and submit a report?  This will allow us to see the exact error message that you're getting from your tool (among other things) and we should be able to offer some advice.

Regarding having bowtie2 already run on the command line outside of Galaxy, if you'd like to just upload those mapped reads to Galaxy and start your analysis in Galaxy there, you definitely could.

ADD COMMENTlink written 3.1 years ago by Dannon Baker3.7k

Dear Dannon,

Thank you so much for writing me back about my galaxy questions. I am using the University of Florida's galaxy instance. I had also written to my local galaxy support folks at the University of Florida and they found that the SAM files I was using did not have the right headers. I am going to fix with their help and try re-run the commands. Here are my errors for the commands I am trying to rerun. I thought I would share this with you in case I am having other problems with running my galaxy commands beyond the Sam file headers.

Thanks, Becky Lazensky

ADD REPLYlink written 3.1 years ago by beckylazensky0
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