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  • bs-seq in colorspace

    there is some discussion of this in another thread.

    i'm considering doing the following for bs-treated reads in colorspace:

    + convert C to T in reference (and for reverse-complement of reference)
    + for reads:
    - convert to nucleotide space
    - convert C to T
    - convert back to colorspace

    + use aligner (bowtie/bfast) to do mappings
    + ... do usual stuff to calculate per-base methylation


    what's wrong with this approach? or how could it be improved for colorspace? is this valid with a subset of reads, so that i could align some this way and then use, for example SOCS (which is very computationally intensive) for unmapped reads?

    thanks for any ideas.

  • #2
    Converting to nucleotide space is generally a bad idea. Converting back will sometimes alleviate this problem, but I don't think that's true in this case.

    A single mismatch in colorspace will change the identifications for all subsequent positions. That will mean:
    1) some bases that were C were not called correctly, so not changed to T. The colorspace representation doesn't change even though it should.
    2) some bases that weren't C were incorrectly called as C, so they were changed to T. The colorspace representation changes even though it shouldn't.

    It might be possible to find color transitions that imply a base is C and change those to the corresponding transition for base T, but I think that would miss a few cases, and hence a lot of reads.

    I could be wrong on my assessment here, but it seems like it would produce quite a few false positives and false negatives. I don't have any better ideas, though.

    It seems that the new ECC stuff from ABI would alleviate a lot of this issue, as it allows trivial conversion to base-space. That assumes, though, that you haven't run the samples yet and have available either the equipment or a collaborator/service that has the latest equipment, as it doesn't work on SOLiD 4 or older.

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