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Old 05-08-2013, 12:33 PM   #1
Eric Fournier
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Location: Quebec, Canada

Join Date: Jul 2011
Posts: 21
Default Normalizing with ERCC spike-in

Hello everyone,

I have RNA-seq libraries prepared from 10 different stages of embryonic development (3 replicates per stage), with each library constructed using the same number of embryos. The amount of total RNA should be variable between stages and follow a known pattern. Thus, to be able to compare "absolute quantities" of RNAs, we spiked each of the libraries with ERCC controls after RNA extraction, but prior to any other processing step.

I've aligned the sequencing results using TopHat2, and constructed transcripts using cufflinks. However, now that I'm getting to the "normalize using ERCC" steps, I am a unsure on how to proceed.

My first instinct would be to do a regression of the ERCC's FPKMs against their known concentration for each of the libraries, then report all of the other transcripts' FPKM against that curve. However, given that FPKMs are already a normalized value, is this still a good idea?

Furthermore, going from FPKM to whichever measure I obtain will make it impossible to use standard RNA-seq comparison tools, such as cuffdiff. Would there be another kind of normalization which would be more "standard" or more sensible?

Thank you for any help,
-Eric Fournier
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