Seqanswers Leaderboard Ad

Collapse

Announcement

Collapse
No announcement yet.
X
 
  • Filter
  • Time
  • Show
Clear All
new posts

  • BWA with no mismatches - n=0?

    Hello,
    We have a sample containing several bacterial species and we want to uniquely map RNA-seq reads to the genomes of each of our organisms to get the expression patterns of each organism separately. We tried to use BWA (implemented in "Galaxy", poor GUI-users that we are) with the “edit distance” (aln -n in the command line version) set to 0 but none of the reads were mapped (all had the SAM tag set to “4’). This is an artifact since running BLAST with some of the sequences showed that they have 100% identity to one of our genomes and not any others, so they should map uniquely.

    Does the "aln -n" option really determine the number of mismatches? Any ideas why BWA will not run well using –n=0?
    Thanks
    Daniel

  • #2
    -n 0 should work at least I tried that before. How long is your reads? When you don't do -n 0, did you get any good alignment, what percentage?

    Best,

    Dong

    Comment


    • #3
      Hi Dong,
      Thanks for your reply. Our reads are 40bp long after QC. When we allow n=1 we get many millions of mapped reads(~90% of the reads map well), and increasing n from 1-5 increases the number of mapped reads but not by very much, so everything is looking OK - apart from when we use n=0.
      Are there any other parameters we should be changing together with n?
      Daniel

      Comment


      • #4
        How about you try to run BWA directly? See what happens.

        Comment


        • #5
          We might try running it directly but part of the idea is to implement BWA as part of a workflow in Galaxy (we are poor "wet" biologists trying to survive in a world of millions of reads...).

          Comment


          • #6
            No no I wasn't suggesting you always do this, this is only to troubleshooting where the problem might be, by running directly with BWA you can figure out if this is something to do with BWA or it's GALAXY.

            Comment


            • #7
              Hi Dong - we don't have BWA installed anywhere as a stand-alone yet. Do you have a standalone BWA installed? If so, if I send you a small file with some sequences and the appropriate genome, is there any chance you can try mapping them with n=0 and send me the results?
              Daniel

              Comment


              • #8
                Ok. You can leave your file over dropbox.

                Comment


                • #9
                  Thanks! What email should I send the dropbox invitation to?

                  Comment


                  • #10
                    You can send private msg or email via seqanswers in fact.

                    Comment


                    • #11
                      Thanks Dong - I just sent a private email with the data (we will follow up with the results on this thread). Daniel

                      Comment


                      • #12
                        Feedback from Galaxy team

                        Hi Daniel -

                        As we posted back on the galaxy-user list, the exact problem you were having was a combination of an issue with the BWA binary itself and the design of the Galaxy BWA wrappper.

                        The original reply and current work-around is available here:


                        We will be addressing this as a priority. Anyone interested in tracking the progress can follow our Trello development ticket here:
                        Organize anything, together. Trello is a collaboration tool that organizes your projects into boards. In one glance, know what's being worked on, who's working on what, and where something is in a process.


                        You uncovered what we believe is a novel issue. Perhaps not commonly run into during command-line usage, but still unexpected. Thanks again for reporting the problem so clearly,

                        Jen
                        Galaxy team
                        Jennifer Hillman Jackson
                        http://usegalaxy.org

                        Comment

                        Latest Articles

                        Collapse

                        • seqadmin
                          Strategies for Sequencing Challenging Samples
                          by seqadmin


                          Despite advancements in sequencing platforms and related sample preparation technologies, certain sample types continue to present significant challenges that can compromise sequencing results. Pedro Echave, Senior Manager of the Global Business Segment at Revvity, explained that the success of a sequencing experiment ultimately depends on the amount and integrity of the nucleic acid template (RNA or DNA) obtained from a sample. “The better the quality of the nucleic acid isolated...
                          03-22-2024, 06:39 AM
                        • seqadmin
                          Techniques and Challenges in Conservation Genomics
                          by seqadmin



                          The field of conservation genomics centers on applying genomics technologies in support of conservation efforts and the preservation of biodiversity. This article features interviews with two researchers who showcase their innovative work and highlight the current state and future of conservation genomics.

                          Avian Conservation
                          Matthew DeSaix, a recent doctoral graduate from Kristen Ruegg’s lab at The University of Colorado, shared that most of his research...
                          03-08-2024, 10:41 AM

                        ad_right_rmr

                        Collapse

                        News

                        Collapse

                        Topics Statistics Last Post
                        Started by seqadmin, Yesterday, 06:37 PM
                        0 responses
                        10 views
                        0 likes
                        Last Post seqadmin  
                        Started by seqadmin, Yesterday, 06:07 PM
                        0 responses
                        9 views
                        0 likes
                        Last Post seqadmin  
                        Started by seqadmin, 03-22-2024, 10:03 AM
                        0 responses
                        49 views
                        0 likes
                        Last Post seqadmin  
                        Started by seqadmin, 03-21-2024, 07:32 AM
                        0 responses
                        67 views
                        0 likes
                        Last Post seqadmin  
                        Working...
                        X