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  • #16
    Originally posted by cliffbeall View Post
    I am fairly sure fr-firststrand means read one (aka forward read or fr) corresponds to the reference genome sequence (firststrand). fr-secondstrand means read one corresponds to the reverse complement (secondstrand) of the reference genome. FR and RF would be a similar concept - but they are just making a shorthand where if the read one or forward read corresponds to the reference strand it maps to the left of the reverse read on a linear representation of the genome (FR) and if it corresponds to the reverse complement of the reference it maps to the right of the reverse (RF).

    I hope that makes sense -- you might need to go back to DNA/genome structure and how Illumina works to really understand the above.
    I'm afraid that this is incorrect.

    First 'fr' does not mean 'forward read'. What it means is the the two reads are arranged in the FR (Foward/Reverse) as opposed to the RF (Reverse/Forward) orientation. FR means that the read pairs are oriented with the 3' end point toward each other when aligned to the reference and RF means they are pointing away from each other.

    Code:
    FR:
    -------------------------------------------------------------------------
       ----------------->                              <-------------------
    
    RF:
    -------------------------------------------------------------------------
       <-----------------                               ------------------->
    Firststrand means that read #1 matches the first strand of cDNA generated during conversion of the single stranded mRNA to ds-cDNA. In other words the R1 is the reverse complement of the mRNA. Note that all of these reference landmarks are relative to transcript, not the genome. For Illumina strand specific, paired end mRNA-Seq reads the orientation of the reads relative to their original RNA is:

    Code:
      mRNA 5'-----------------------------------------------------------AAAAAAAAAAAA
                       R2 ------------>                <------------ R1

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    • #17
      Looks like Jim Robinson had it backwards for the color legends. Actually

      red=positive,
      blue=negative

      He has clarified here:

      Comment


      • #18
        Originally posted by sowmyai View Post
        Looks like Jim Robinson had it backwards for the color legends. Actually

        red=positive,
        blue=negative

        He has clarified here:
        https://groups.google.com/forum/#!to...lp/YiVzwnUuOZM
        I always thought that blue=positive and red=negative. See screen shots of my data in IGV.

        I used Illumina dUTP-type stranded library prep and fr-firststrand with Tophat2. I find several places that say dUTP libraries should use fr-firststrand with Tophat, for example: http://onetipperday.sterding.com/201...pe-to-use.html


        Is anyone able to clear this up?

        Thanks
        Attached Files

        Comment


        • #19
          Tablet has a "read concordance" mode for viewing strand direction on reads. Both pairs of reads that are consistent with a mapped direction following the reference sequence are coloured green (customisable), and reads that are consistent with the reverse complement of the reference sequence are coloured red.

          Comment

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