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  • TopHat and Multireads - what is happening under the hood?

    Hi all -

    I have had success so far with TopHat, and it is a great tool!

    My RNA-seq workflow is to run TopHat and then Cufflinks. Recently, I noticed that some of the transcripts that are produced are "false positives" due to multi-mapped reads. In otherwords, the vast majority of coverage (RPKM) in those transcripts due to reads that map to multiple locations.

    I could eliminate all of these false positives by setting TopHat's (-g/--max-multihits) option to '1', but I would rather not do this because it would sacrifice sensitivity in exchange for guaranteed uniqueness.

    Ideally, I want to be able to know some statistics about how many of the reads in each transcript came from multi-mapping vs. uniquely mapping reads. Is there any information in the 'accepted_hits.sam' file about this? What about in any of the other temporary files that TopHat generates.

    How does everyone else handle multi-mapping reads and TopHat?

    Also, in the SAM output file, I noticed that the mate pair reference is always set to '=' even if the two mates map to different references.

    Finally, would it be possible to add the 'insert size' parameter to the accepted_hits.sam file?

    Best regards, and thanks again!

  • #2
    Originally posted by choy View Post
    Hi all -
    Also, in the SAM output file, I noticed that the mate pair reference is always set to '=' even if the two mates map to different references.

    Finally, would it be possible to add the 'insert size' parameter to the accepted_hits.sam file?

    Best regards, and thanks again!
    In tophat manual there is already an option for the expected inner distance between mate pairs. I think, it would help you.

    -r/--mate-inner-dist <int> This is the expected (mean) inner distance between mate pairs. For, example, for paired end runs with fragments selected at 300bp, where each end is 50bp, you should set -r to be 200. There is no default, and this parameter is required for paired end runs.

    Best
    Meryem

    Comment


    • #3
      Originally posted by MerFer View Post
      In tophat manual there is already an option for the expected inner distance between mate pairs. I think, it would help you.

      -r/--mate-inner-dist <int> This is the expected (mean) inner distance between mate pairs. For, example, for paired end runs with fragments selected at 300bp, where each end is 50bp, you should set -r to be 200. There is no default, and this parameter is required for paired end runs.

      Best
      Meryem
      Thanks Meryem. I am actually setting this parameter already. However, even though we pass the expected inner distance to TopHat, each individual mate pair will have its own insert size when mapped.

      I suppose that since TopHat maps to the human genome, it is not possible to approximate the actually insert size until transcript assembly is complete.

      Best regards,

      Comment

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