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  • Forward strand bias?

    Hi all,
    this may be something known... anyway, tags coming from Illumina sequencing method (GA II here) seem to map preferentially on forward strand... I haven't check if there is a statistically significant variation, but apparently tags aligned on forward strand are 2-5% more (in various ChIP-seq and also in genome resequencing).
    I wonder which could be the source of this variation... The library prep? The cluster generation? The aligner? None of these steps makes sense to me...

    d

  • #2
    ?! strange indeed!
    statistical significance may be an important point here.. is the bias constant across chromosomes?
    s.

    Comment


    • #3
      What aligner are you using? Probably you could use a different one and see what happens to the bias
      --
      bioinfosm

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      • #4
        I'd also suggest taking your references and reverse complementing them. Then repeat the alignment on the RC reference and see what happens. If this is just a software artefact, then you should see more reads on the new forward strand (i.e. what was the reverse strand in the original reference).

        Comment


        • #5
          My bet would be a single genomic position (on the forward strand) that gets a gazillion of copies of a single read (generated by a contaminant, an amplification artifact, an incorrect filter, or something else).

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          • #6
            Originally posted by dawe View Post
            Hi all,
            this may be something known... anyway, tags coming from Illumina sequencing method (GA II here) seem to map preferentially on forward strand... I haven't check if there is a statistically significant variation, but apparently tags aligned on forward strand are 2-5% more (in various ChIP-seq and also in genome resequencing).
            I wonder which could be the source of this variation... The library prep? The cluster generation? The aligner? None of these steps makes sense to me...

            d
            Hi,
            any news regarding this issue?
            thanks,
            s.

            Comment


            • #7
              Originally posted by steven View Post
              Hi,
              any news regarding this issue?
              thanks,
              s.
              Not yet... I've been busy on other issue but I will investigate this further...

              Comment

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