[BioC] questions about DESeq package: estimateSizeFactors() function

[Simon Anders]

Dear Sara

On 09/20/2011 03:39 PM, Sara Nadaf wrote:
> I am using DESeq package for doing the differentially expressed analysis of
> miRNA in two conditions (case and control) without replicate.
>
> When I run estimateSizeFactors() function, the result of sizeFactors(cds) is
> 1 and 1 for both conditions.
>
> 1- Is it normal to have same sizeFactors for both conditions?

Getting exactly 1 tends to happen if your count values are all very low. 
To check whether the size factors are appropriate, make an MvA plot 
comparing the two samples

    plot( ( counts(cds)[,1] + counts(cds)[,2] )/2,
       counts(cds)[,2] / counts(cds)[,1], log="xy", pch="." )

and add a horizontal line to show the scaling constant

    abline( h = sizeFactors(cds)[1] / sizeFactors(cds)[2] )

Now check whether the horizontal line dissects the bulk of points nicely 
in the middle, especially at the larger count values.

> 2- This function use for normalization of raw data, right? Or I should
> normalize the data before using the package?

No, raw values are correct.

   Simon