User Reference Vs. Built-In Index

Question: User Reference Vs. Built-In Index

8.6 years ago by

Viren Patel • 30 wrote:

Hi. I created a workflow to map IGA reads using bowtie and generate a pileup at the end. The workflow is FASTQ Groomer -> Map with Bowtie for Illumina -> SAM-to-BAM -> Generate pileup. I ran the workflow successfully using the built-in mm9 index as reference for both bowtie and the pileup generation. Then I changed the workflow to use a reference file I uploaded (fasta format) and now I get an error on the pileup generation step: An error occurred running this job: /The output file is empty. Your input file may have had no matches, or there may be an error with your input file or settings./ The prior SAM-to-BAM step shows a sizeable BAM file being generated. So I am wondering if the Generate pileup tool requires the reference in a special format. V. Patel Dept. of Human Genetics Emory University School of Medicine

alignment bowtie • 1.7k views

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modified 8.5 years ago • written 8.6 years ago by Viren Patel • 30

8.6 years ago by

Rick Westerman • 30

Rick Westerman • 30 wrote:

I am a new Galaxy user but it seems to me that the problem lies with using a built-in index vs. your own fasta file that needs an index built for it. I suspect that the parameters for the workflow are set to use built-in indexes. My suggestion is to manually run the steps instead of relying on the workflow. -- Rick Westerman westerman@purdue.edu Bioinformatics specialist at the Genomics Facility. Phone: (765) 494-0505 FAX: (765) 496-7255 Department of Horticulture and Landscape Architecture 625 Agriculture Mall Drive West Lafayette, IN 47907-2010 Physically located in room S049, WSLR building

ADD COMMENT • link written 8.6 years ago by Rick Westerman • 30

I changed the workflow to use my own uploaded reference file, not the built-in index, but judging from the nomenclature I suspect I may have to process (index?) the reference file first into a format usable by the pileup generation tool. However I do not see how I can do that.

ADD REPLY • link written 8.6 years ago by Viren Patel • 30

Galaxy should do this automatically. At least it did it for me yesterday (admittedly using SOLiD data instead of Illumina. I still recommend manually running each step instead of relying on a workflow. -- Rick Westerman westerman@purdue.edu Bioinformatics specialist at the Genomics Facility. Phone: (765) 494-0505 FAX: (765) 496-7255 Department of Horticulture and Landscape Architecture 625 Agriculture Mall Drive West Lafayette, IN 47907-2010 Physically located in room S049, WSLR building

ADD REPLY • link written 8.6 years ago by Rick Westerman • 30

The indexing should be automatic. We'll need to look at it in more detail. -- jt (composed on my phone)

ADD REPLY • link written 8.6 years ago by James Taylor • 320

Hi, You will not have to generate the index separately (you actually can't do this--if you're not using one of the built-in indexes, you have to supply a fasta file to be indexed on each mapping run). Changing to an uploaded reference should work within your workflow, just as it should work if you ran each step outside of the workflow. I can look into what's going wrong here if you will share both your workflow and your history with me (kpvincent@bx.psu.edu). To share your workflow: From the workflow menu, click on the arrow next to your workflow and select Share or Publish. Then click the Share with a user button near the bottom of the page and enter my email address in the following box. To share your history: From the history pane, click on the Options button in the upper right and select Share or Publish. Then click the Share with a user button and enter my email address in the following box. Regards, Kelly

ADD REPLY • link written 8.6 years ago by Kelly Vincent • 340

8.5 years ago by

Viren Patel • 30

Viren Patel • 30 wrote:

Hi Kelly. I just wanted to follow-up on this issue. I did some more tests and it seems that the problem may be because the fragement headers in my custom reference multi-fasta contain the coordinates of the fragment in the form chr:start-stop. Upon mapping with bowtie, the resultant SAM file contains the headers (as expected). But the BAM-to-pileup tool does not play well with such headers. It seems the BAM-to-pileup tool expects headers in only chr format. Since there does not appear to be a tool on galaxy to convert chr:start-stop to chr format in a SAM file (note: this also requires modification of the next column in the SAM file), the solution at this point is to download the SAM file, locally apply the fix, re-upload the SAM file and run the BAM-to-pileup tool with a built-in index (mm9 in my case). Even at this step, I still can't use my custom reference for the pileup because it contains headers in chr:start-stop format. Thoughts?

ADD COMMENT • link written 8.5 years ago by Viren Patel • 30

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