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  • more 'C's than 'G's!!

    Dear Forum,

    Does anyone have information about a G vs C bias? I am analysing 1000 genomes data based on raw fastq files ~5Gb in size. For other reasons I counted the occurrence of each base and found, to my surprise, that 'C's outnumber 'G's by about 10% (though the size of the bias varies between sequencing centres). To guard against artefacts, I then counted G-C frequencies for each base position in each read (I only look at forward runs, so have bases 1 to 100). I see that the G vs C bias varies with base number, being very variable for the first and last 5 bases and otherwise generally rising (= more 'C's) along the read.

    Is this a well-known phenomenon? Any thoughts gratefully received!

    Cheers
    Bill

  • #2
    Sounds like a calibration problem. I have not seen this in our data.

    The variability at the ends, though, I have seen. This is sometimes due to adapter contamination, and sometimes due to the fact that base-calling uses information about adjacent bases, which is absent at the tips.
    Last edited by Brian Bushnell; 03-12-2014, 09:37 AM.

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    • #3
      have you looked specifically at C / G imbalance?

      Dear Brian,

      Thanks. The pattern seems remarkably pervasive - I have looked at data from SC, BGI and BCM (all show quite strongly in ~75 reads, each 5Gb) and WUGSC (less strong effect). There does also seem somewhat of a population effect, though I'd need to look at more samples to be sure. If you take an index 2*(C - G) / (C + G), where C and G are the numbers of 'C's and 'G's, this typically rises from 0.08 to o.12 between based 5 and 95.

      Cheers
      Bill

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      • #4
        Originally posted by Bill Amos View Post
        I then counted G-C frequencies for each base position in each read (I only look at forward runs, so have bases 1 to 100).
        Hi- Isn't this the information reported by FastQC, module "Per base sequence content"?

        I looked at the FastQC report for some data we have (ChIP-Seq, FAIRE-Seq) and I don't see much difference between C and G after the first 5-10bp. Although the first bases are indeed very biased but I think this is due to the non-so-random fragmentation.
        Attached Files

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        • #5
          FastQC

          Yes, FastQC does present the relevant data, though not as a ratio / difference. Slight (G 5% down, C 5% up) might not show up very clearly, either in this plot or, of course, as GC content, particularly if the first few bases are wayward. I guess this sort of bias 'comes out in the wash' when the reads are aligned, but I'm trying to analyse data without any of the biases that alignment can introduce!

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