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  • Size variation for multiplexing

    Hi friends,

    I prepared mulitplexing libraries for ChIP-Seq of a histone marker in different cell types. I used sonication as the chromatin shearing method and used the Truseq adaptors for generating the libraries. However, the size distribution of the libraries I made varied from one to the other with a maximum difference of about 40bp (bioanalyzer image attached). Could anyone here give me some suggestion as whether this would affect the outcome of the experiment? What would be a decent size difference to expect when doing multiplexing? I also found that the input samples tend to have more such variations.

    Many thanks.

    PingClick image for larger version

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    Last edited by Snf5; 09-09-2012, 06:33 AM. Reason: I found that my pdf attachment may be blocked

  • #2
    Variation in the fragment size between different transcription factors or histone markers is normal and I don't think I've ever seen a problem as a result of the magnitude change that you observe.

    Your case is a little different, since it's all the same marker. I assume you're testing a set of conditions over which you expect to see some changes (more or less of the marker, or different targets), so it's not surprising to see some change in the size fragments you get. If these were my samples, I'd probably go ahead and sequence them. However, I'd do some extra analysis to see whether parameters in the analysis software correlate with the fragment sizes- eg, does setting a smaller or larger window for enrichment matter? If so, you'll need to think about how to account for that if you're doing overlap statistics between conditions.

    Alex

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    • #3
      Hi Alex,

      Thank you very much for your advice. Really appreciated it.

      Ping

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