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I'm working on an RNA-Seq project and I am trying to figure out library normalization. I'm aware of using the geometric means (e.g. cuffdiff) of the fpkm for the normalization.

However, I was wondering why people don't add some unique, known RNA sequence of known concentration into their sample prior to amplification. Then after sequencing you would have some known measure by which to normalize your fpkm. I would think this would also be a way to minimize batch effects.

Is there a technical reason why this isn't done?

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    $\begingroup$ People do do that. Google "ERCC spike-in probes" $\endgroup$ – swbarnes2 Sep 23 '16 at 20:09

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