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  • shocker8786
    Member
    • Jan 2013
    • 28

    Align paired and unpaired reads with Tophat

    I have a stranded PE RNAseq data set that I want to align with tophat using the --library-type fr-firststrand option. After adaptor trimming I end up with my 4 files:

    paired_1
    paired_2
    unpaired_1
    unpaired_2

    Is there a way to align these so I do not loose the strand information for the unpaired reads? When I run tophat with the files listed like this:

    paired_1,unpaired_1 paired_2,unpaired_2

    It seems to want to try and align the two unpaired files as paired files. If I combine the two unpaired files and run tophat with the files listed like this:

    paired_1 paired_2,unpaired

    It recognizes the last file as unpaired. But am I loosing my strand specificity by aligning this way? Thanks.
  • dpryan
    Devon Ryan
    • Jul 2011
    • 3478

    #2
    Inputting the files like:
    Code:
    paired_1,unpaired_1 paired_2,unpaired_2
    Will result in "unpaired_1" and "unpaired_2" being treated as paired, which is the opposite of what you want. To keep the strandedness correct you'll need to run things twice. Firstly using "paired_1,unpaired_1 paired_2" with library-type set to fr-firststrand and then just "unpaired_2" by itself with "fr-secondstrand". I should note that aligning unpaired_2 is usually not worthwhile (the reads are often crap), but perhaps you'll get luckier than I have with that.

    Comment

    • shocker8786
      Member
      • Jan 2013
      • 28

      #3
      Thank you very much for your response, that makes a lot of sense.

      Comment

      • natar210@gmail.com
        Junior Member
        • Jun 2013
        • 2

        #4
        On this note ..I Have a question. I am doing PE RNA-Seq analysis of mouse data. My read length is 90bp and fragment size is 127 bp which means I have overlapping reads. I used Flash and found that not all reads overlap.

        I have a file with merged reads which overlaps and also two files with non overlap reads. basically three fastq files.

        How do I go about running Tophat with this and I am not really sure hot to calculate the mean inner mate distance and sd??

        Has anyone come across this situation???

        Comment

        • dpryan
          Devon Ryan
          • Jul 2011
          • 3478

          #5
          There's no need to run Flash on them, just use tophat2 and bowtie2 instead of bowtie1.

          For the mean inner distance, just try 0 and see if that produces acceptable results (I recall reading that tophat re-estimates the insert length as it runs, though I can't say I've ever checked if that's correct).

          Comment

          • natar210@gmail.com
            Junior Member
            • Jun 2013
            • 2

            #6
            Thanks alot Ryan... I will try that.

            Comment

            • NicoBxl
              not just another member
              • Aug 2010
              • 264

              #7
              or just use STAR that do not need to specify inner distance. and also is much faster for the same ( even better ) results

              Comment

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