Deseq Sizefactors Help

Question

[Sara]

Hello,

I try to do the diff analysis by DESeq on two samples coming from two different condition without replicate

I run the script of DESeq and when I give the library size of my sample, the size factor is 1 for both conditions

libsizes <- c( Cond1 =80040653, Cond2 =360265740)
cds=estimateSizeFactors(cds)
sizeFactors(cds)

    Cond1 Cond2 
       1    1

I don't understand this estimation?

when I don't give the libsizes, it become

 sizeFactors(cds)
     Cond1 Cond2 
1.7320508 0.5773503

Could you please explain for me this issue?

Thanks in advance Sara

[Vitis]

Probably you assigned sizes first and then did the estimate, which may just scale your assigned sizes back to 1. I think the right way is to estimate the factor directly from the 'cds' object without assigning any libsizes.

[Sara]

Hi Jeremy thanks for your comment could u please tell me what did this sizeFactors function? is it for normalization of data (scale normalization)?

Answer 1

[Damian Kao]

The author of DESeq wrote a post on SeqAnswers a while back about how the size factors thing work in DESeq. I'll try to find the post, but basically it normalizes the datasets by:

-Take the geometric mean of each condition for a gene and use that as the reference expression data set.

-For each condition, get a list of quotients of each gene expression value to its reference expression.

-The median of each condition quotient list is the normalization factor for that data set.

edit*

Here is the post: http://seqanswers.com/forums/showpost.php?p=16468&postcount=13

Answer 2

[Jeremy Leipzig]

I don't see where in the documentation it says libsizes is a magic word.

Either use the estimateSizeFactors to use your count data as the source of the estimate, or set it manually using sizeFactors.

Now, why are there so many Sara's?

[Pasta]

Because Sara is a very popular name amongst bioinformatician LOL

[Istvan Albert]

could be some OpenID flaking - good catch - merged the Sara-s - There Can Be Only One