mark.robinson at imls.uzh.ch
Wed Mar 28 16:56:29 CEST 2012
As I mentioned (in our offline conversation yesterday), I don't know of an obvious way to normalize against multiple samples. But, as I mentioned, I'm not convinced that it is necessary, or advisable. Maybe you can convince us why you want to do this? One could argue that even between biological replicates, variations (even artifacts) occur that would make the scaling factors different than 1.
On 27.03.2012, at 03:48, Anand K S Rao wrote:
> Naive question:
> For calcNormFactors(), while using method="TMM", for normalization, can
> multiple independent biological replicates be used to normalize against?
> Help on calcNormFactors in edgeR seems to indicate using only one reference
> library / column (copied and pasted below).
> Could someone please clarify?
> calcNormFactors(object, method=c("TMM","RLE","upperquartile"),
> refColumn = NULL, logratioTrim = .3, sumTrim = 0.05, doWeighting=TRUE,
> Acutoff=-1e10, p=0.75)
> object: either a Œmatrix‚ of raw (read) counts or a ŒDGEList‚ object
> method: method to use to calculate the scale factors
> refColumn: column to use as reference, only used when Œmethod="TMM"‚
> logratioTrim: amount of trim to use on log-ratios ("M" values), only
> used when Œmethod="TMM"‚
> sumTrim: amount of trim to use on the combined absolute levels ("A"
> values), only used when Œmethod="TMM"‚
> doWeighting: logical, whether to compute (asymptotic binomial
> precision) weights, only used when Œmethod="TMM"‚
> Acutoff: cutoff on "A" values to use before trimming, only used when
> p: percentile (between 0 and 1) used to compute scale factors
> from, only used when Œmethod="upperquartile"‚
> Œmethod="TMM"‚ is the weighted trimmed mean of M-values (to the
> reference) proposed by Robinson and Oshlack (2010), where the
> weights are from the delta method on Binomial data. If
> ŒrefColumn‚ is unspecified, the library whose upper quartile is
> closest to the mean upper quartile is used.
> Anand K.S. Rao
> PhD candidate
> Plant Biology Graduate Group
> Indian Graduate Student Association @ UCDavis
> University of California at Davis
> Davis, CA - 95616 USA
> (translate THAT!)
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Prof. Dr. Mark Robinson
Institute of Molecular Life Sciences
University of Zurich
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