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  • Tophat's bowtie parameters

    Hi folks,

    I am using tophat in my RNA-seq of a high polymorphic genome. I think Tophat calls bowtie with '-v 2', but I found many reads with more than 2 mismatches were mapped to the genome. I took one as the test, it has 5 mismatches with good phred quality scores:

    13:C>T,27:C>A,38:T>C,57:G>A,64:C>A

    Using -v 3, or default -n 2 -l 28 -e 70 cannot map it, but using -n 2 -l 28 -e 150 can map it.

    Why can tophat map these kind of reads?

    Thanks!

  • #2
    I also have a quite similar question: How to set the other bowtie parameters in tophat(such as "-a --best --strata")?

    Comment


    • #3
      Originally posted by Gangcai View Post
      I also have a quite similar question: How to set the other bowtie parameters in tophat(such as "-a --best --strata")?
      I did it just by editing the python script of tophat.
      Xi Wang

      Comment


      • #4
        Originally posted by Xi Wang View Post
        I did it just by editing the python script of tophat.
        How exactly did you do this? Which line and what is the name of the script?

        I appreciate your help in advance. Thanks !

        Comment


        • #5
          you can modify the tophat bin file

          The file you should modify is tophat-1.0.13/bin/tophat. I have an example file in the attached file for my question(add --best, --strata and --seedmms)(the modified line have #by gangcai)

          After modification, you can run tophat like this:

          $tophat --best --strata --seedmms 3 ......

          Originally posted by thinkRNA View Post
          How exactly did you do this? Which line and what is the name of the script?

          I appreciate your help in advance. Thanks !
          Attached Files

          Comment


          • #6
            Originally posted by Gangcai View Post
            The file you should modify is tophat-1.0.13/bin/tophat. I have an example file in the attached file for my question(add --best, --strata and --seedmms)(the modified line have #by gangcai)

            After modification, you can run tophat like this:

            $tophat --best --strata --seedmms 3 ......
            Great! I think tophat developers should also take those options into consideration.
            Xi Wang

            Comment


            • #7
              I agree, one of the strengths of BOWTIE is being able to modify the way it works via its options- but all of this customizability is lost when using TOPHAT..

              Comment


              • #8
                help with Tophat's bowtie parameters

                Originally posted by Gangcai View Post
                The file you should modify is tophat-1.0.13/bin/tophat. I have an example file in the attached file for my question(add --best, --strata and --seedmms)(the modified line have #by gangcai)

                After modification, you can run tophat like this:

                $tophat --best --strata --seedmms 3 ......

                Hi
                If it is not too much trouble, can you post a tophat file with modification to the the latest version (1.2). My data maps better when I set my own -n/--seedmms and -l/--seedlen parameters.
                As you can tell, I don't know how to write scripts.

                Thank you in advance

                Joseph Dhahbi

                Comment


                • #9
                  Originally posted by joseph View Post
                  Hi
                  If it is not too much trouble, can you post a tophat file with modification to the the latest version (1.2). My data maps better when I set my own -n/--seedmms and -l/--seedlen parameters.
                  As you can tell, I don't know how to write scripts.

                  Thank you in advance

                  Joseph Dhahbi
                  I think the authors of Tophat don't want users to run Tophat in this way. One of the reasons should be that those parameters also apply to segment mapping in Tophat. A segment is an arbitrary part of a read, not necessarily from the left end of a read. So the seed region of a segment could be different from the seed of a whole read. This may cause some biases, I guess.
                  Xi Wang

                  Comment


                  • #10
                    TopHat source code modification to include --best

                    Hi all,

                    I want to use TopHat for my mRNA Seq analysis, but I am unable to include --best..

                    I tried modifying the code to include best (similar to what's given above), but I was not successful..

                    Could someone please provide a modification in the latest version (1.3.1)? *I wish tophat did not make it impossible to edit bowtie options on the command line*

                    Comment


                    • #11
                      i have another problem about tophat.
                      i mapped 90bp pair-end reads twice with tophat of different versions with same arguments -g 10 -r 20 --solexa1.3-quals .The default segment-mismatch is 2. in the otput of the older version(1.0.12),the minimum match length is 75bp,why?. in the output of the latest version(1.3.1) it is mostly 90bp, is it because the -v 2 ?
                      is there anyone facing the same problem?

                      Comment


                      • #12
                        Shrutii,
                        U can try a different approach... Might work... If u have already run tophat... there is a log folder in the output directory. Open run.log file. It lists the sequence of commands tophat executes... I think there are 29 steps totally. U can modify the steps in which bowtie is called and give the --best flag... and then run all the commands as a bash script but as Xi Wang mentioned above, I too feel Cole or the other authors of Tophat do not want users to run it this way. I might be wrong tho...

                        Dinesh

                        Originally posted by shrutiisarda View Post
                        Hi all,

                        I want to use TopHat for my mRNA Seq analysis, but I am unable to include --best..

                        I tried modifying the code to include best (similar to what's given above), but I was not successful..

                        Could someone please provide a modification in the latest version (1.3.1)? *I wish tophat did not make it impossible to edit bowtie options on the command line*

                        Comment

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