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  • WGBS - higher levels of non-CG methylation with non-directional libraries?

    EDIT: This has been expanded on and cross-posted at Biostars. I'll update both posts with responses from the other site:

    I've been analysing some WGBS libraries for a couple of different insect species, using Bismark for alignment. For context in most insects methylation levels are low to begin with and non-CG methylation is often considered to be noise.

    I have both directional and non-directional libraries for each species, with alignment performed using the non-directional parameter in Bismark for the latter. I've noticed a pattern of non-directional libraries having higher levels of non-CG methylation (as estimated by Bismark's splitting report generated during methylation extraction). Examples as follows from this report - same species, both control groups:

    Directional:

    Final Cytosine Methylation Report
    =================================
    Total number of C's analysed: 1441325271

    Total methylated C's in CpG context: 10947321
    Total methylated C's in CHG context: 1518883
    Total methylated C's in CHH context: 7907753

    Total C to T conversions in CpG context: 260501288
    Total C to T conversions in CHG context: 203098923
    Total C to T conversions in CHH context: 957351103

    C methylated in CpG context: 4.0%
    C methylated in CHG context: 0.7%
    C methylated in CHH context: 0.8%
    Non-directional:

    Final Cytosine Methylation Report
    =================================
    Total number of C's analysed: 1671579979

    Total methylated C's in CpG context: 15071917
    Total methylated C's in CHG context: 3393242
    Total methylated C's in CHH context: 17650398

    Total C to T conversions in CpG context: 360463445
    Total C to T conversions in CHG context: 264082345
    Total C to T conversions in CHH context: 1010918632

    C methylated in CpG context: 4.0%
    C methylated in CHG context: 1.3%
    C methylated in CHH context: 1.7%
    This pattern holds in all samples and in both species I'm studying. Before I conclude that this is the result of quirks of these specific datasets, I was wondering if anyone was aware of a reason to do with the nature of non-directional libraries as to why the non-CG methylation might be reported at a higher level?
    Last edited by beej; 02-28-2020, 03:21 AM.

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