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  • Removing contaminating sequence from 16s reads

    Our core facility has a customer who wants to look at 16s rDNA on the MiSeq. Their initial PCR has a very faint secondary band about 2x the size of the 16s band they want to sequence. They've got several hundred samples to prep and are unwilling to gel extract to remove the contaminating band. I've told them to try to optimize their PCR as much as possible but it seems almost impossible to get rid of this band entirely. Is there any way to remove the non-16s band bioinformatically after the MiSeq run?

  • #2
    Perhaps.

    It would depend on what the contaminant(s) represents. If the samples are to be multiplexed (and the contaminant is not tagged, then it could be eliminated during de-multiplexing).

    They may have to run at least one batch of samples so someone can access the data.

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    • #3
      "Perhaps" is the scary answer! The libraries are constructed with a two-step PCR so unless they gel extract after their initial PCR, the second PCR will index everything present in the sample. I was thinking that possibly, since there should be some highly conserved regions in their 16s, they could somehow pull out only sequences with that region. My problem is that I am the core lab technician - we make and sequence libraries, but the bioinformatics steps are up to the customers!

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      • #4
        In theory, they can figure out empirically what that stuff in the other band is, and align to that as well as the 16S, and that way, separate out those reads. As the technician, I don't think there's anything you can do to help. You sequence what they give you, if if their protocols are problematic, that's their problem, not yours.

        It might behoove them to run a subset to make sure this contamination isn't a serious issue before proceeding with all their samples.

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