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  • Non-C methylation in Bismark .cov files

    Hi
    I am analyzing RRBS using TrimGalore/Bismark and the methylation extractor, and am noticing quite a few non-C (T, A, and G) methylation signals (maybe 20% compared to C, by eye). Is this a normal technical artifact that has been observed?
    Thanks
    --Bryan

  • #2
    In the case of G, that is a C, just on the other strand. In the case of T or A, that's either an off by 1 error or the sequence you're looking at is different from the sequence against which the aligning was done.

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    • #3
      Thanks for the replay Devon.
      Is the 'off by 1 error' common? What would cause this?
      --Bryan

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      • #4
        Off by 1 errors are quite common (common enough that it has its own wikipedia article). The likely cause is the file using 1-based coordinates while whatever you're using to visualize things assumes it's using 0-based coordinates. I remember this being an issue with some of bismark's output at one point, though I don't know what the status of that is. If you look at those and there's an appropriate C (or G, depending on how things were done) just to the right, then that's what's going on.

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        • #5
          Wouldn't off-by-1 errors more result in 50/50 values than 20%? I'd be happy to have a closer look at whatever problems you are running into, just drop me a line.

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          • #6
            Given the non-random distribution of bases in the genome, probably not.

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