[BioC] LIMMA: Why does eBayes expression differ from observed?

[Edwin Groot]

On Thu, 30 Jul 2009 14:57:43 +0200
 Axel.Klenk at Actelion.Com wrote:
> 
> Dear Edwin,
> 
> first of all, the code you have posted cannot work because it never
> assigns
> the results
> of your computations to any variables. In order to help you, we need
> to
> know at least
> 1) what code you have really run,
---
I shall clean out the comments and sent the code out tomorrow.
Two of the mutants are in chromatin remodelling factors, and are
expected to derepress a significant percentage of the ORFs. One of the
mutants has a relatively trivial effect on the transcriptome. Does that
violate any assumptions in the fitting steps?
---
> 2) your refdesign matrix,
> 3) the values from topTable() and RG.MA() for at least one example
> probe,
> 4) HOW you backcalculated R, G, and FC from topTable(), and
> 5) the obligatory output of sessionInfo() although this doesn't
> really
> sound like a version issue. :-)
> 
---
> sessionInfo()
R version 2.7.1 (2008-06-23) 
i486-pc-linux-gnu 

locale:
LC_CTYPE=en_US.UTF-8;LC_NUMERIC=C;LC_TIME=en_US.UTF-8;LC_COLLATE=en_US.UTF-8;LC_MONETARY=C;LC_MESSAGES=en_US.UTF-8;LC_PAPER=en_US.UTF-8;LC_NAME=C;LC_ADDRESS=C;LC_TELEPHONE=C;LC_MEASUREMENT=en_US.UTF-8;LC_IDENTIFICATION=C

attached base packages:
[1] tools     stats     graphics  grDevices utils     datasets  methods
 
[8] base     

other attached packages:
[1] geneplotter_1.18.0  annotate_1.18.0     xtable_1.5-5       
[4] AnnotationDbi_1.2.2 RSQLite_0.7-1       DBI_0.2-4          
[7] lattice_0.17-10     Biobase_2.0.1       limma_2.14.7       

loaded via a namespace (and not attached):
[1] grid_2.7.1         KernSmooth_2.22-22 RColorBrewer_1.0-2
---

> AFAIK, eBayes() will affect the t, p, and B statistics computed from
> your M
> values but not
> the AveExpr and logFC.
> 
> RG.MA() backcalculates background-adjusted and normalized R and G
> values,
> so I am
> not sure what you mean by "observed" RG -- you don't get back the
> original
> R, Rb, G, Gb
> from an MAList via RG.MA().
> 
> Cheers,
> 
>  - axel
> 
> 
> Axel Klenk
> Research Informatician
> Actelion Pharmaceuticals Ltd / Gewerbestrasse 16 / CH-4123 Allschwil
> /
> Switzerland
> 
> 
> 
>
                                                                      
>     
>              "Edwin Groot"
>                                                 
>              <edwin.groot at biol
>                                             
>              ogie.uni-freiburg
>                                          To 
>              .de>                      bioconductor at stat.math.ethz.ch
>      
>              Sent by:
>                                                   cc 
>              bioconductor-boun
>                                             
>              ces at stat.math.eth
>                                     Subject 
>              z.ch                      [BioC] LIMMA: Why does eBayes
>       
>                                        expression differ from
> observed?    
>
                                                                      
>     
>              30.07.2009 12:38
>                                              
>
                                                                      
>     
>
                                                                      
>     
>
                                                                      
>     
>
                                                                      
>     
> 
> 
> 
> 
> I am getting odd results from a common reference design analysis of
> two-colour data in LIMMA. Previously I had analyzed only
> simple-comparison designs. Hopefully you can help restore credibility
> of Bioconductor to my supervisor.
> Why does the AveExpr and logFC reported in topTable() differ from the
> replicates in my MA object?
> 
> The analysis is a textbook example of comparing a series of mutants
> to
> the wild type. Wild type is always green. The summary is as follows:
> 
> lmfit(MA, refdesign)
> eBayes(fit)
> topTable(fit, coef="mut1")
> #Regenerate the RG from MA
> RG.MA(MA)
> 
> Backcalculating the topTable AveExpr and logFC to green, red and FC
> gives the following expressions as an example:
> WT: 31   mut1: 298   FC: 9.5
> Compare that to the average of 9 WT and 3 mut1 in the RG.MA(MA) list:
> WT: 58   mut1: 611   FC: 10.5
> 
> A survey of other probes gives an over and underestimate of the
> observed RG from 1.5 to 5 times.
> What is the explanation for that?
> What should I troubleshoot, besides looking at the usual BG and FG
> distributions and MA plots?
> 
> Regards,
> Edwin
> ---
> Dr. Edwin Groot, postdoctoral associate
> AG Laux
> Institut fuer Biologie III
> Schaenzlestr. 1
> 79104 Freiburg, Deutschland
> +49 761-2032945
> 
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Dr. Edwin Groot, postdoctoral associate
AG Laux
Institut fuer Biologie III
Schaenzlestr. 1
79104 Freiburg, Deutschland
+49 761-2032945