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  • RNA-seq and GC content bias

    Dear all,

    I am analyzing a dataset of RNA-seq (from Illumina Genome Analyser IIx), and have found some interesting expression pattern trends related to the GC content of genes. After reading the literature, I have noticed that RNA-seq has some biases related to the GC content. Hence, I am not sure of the validity of my inferences.

    So, my question, or questions are:
    -How can I determine if this is a biologically meaningful trend or rather so a bias of the methodology?
    -To what extent does the GC content biases the results of RNA-seq experiments? (the "trends" that I observe are strong)
    -I am working with published RPKM values, and was wondering if I need to recalculate all these values considering GC content biases or if I could just test how much bias is in the data.

    Any clue with any of these questions is highly appreciated!
    Federico

  • #2
    Have a look at the conditional quantile normalization paper from Hansen et al. (http://biostatistics.oxfordjournals..../13/2/204.full). I expect that has much of what you're looking for.

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    • #3
      Many thanks, dpryan

      I am not sure whether I could guess from the paper the extent of the influence of the GC content, but will do some tests with my data to see what can I see

      Fede

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      • #4
        This was also a concern of mine since I work with a GC-rich species, and it is clear from Aird et al, 2011 (Genome Biology 12:R18) that PCR conditions during library prep can non-uniformly amplify transcripts with different GC contents. If you are really seeing systematic biases, I would figure out what you did/had done during library construction (polymerase, buffers, etc.), to know if you can anticipate that you had biased amplification.

        I am not sure this will help your analysis any, but at least if your preparation didn't take this into account, you can write off some of the variability that way.

        Anna

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