[BioC] barley cdf

[James W. MacDonald]

Hi Lev,

Lev Soinov wrote:
> Dear List,
>   Sorry, I attached a wrong sessionInfo() to my last e-mail (below). It should be:
>   > sessionInfo()
> R version 2.4.1 (2006-12-18) 
> i386-pc-mingw32 
>   locale:
> LC_COLLATE=English_United Kingdom.1252;LC_CTYPE=English_United Kingdom.1252;LC_MONETARY=English_United Kingdom.1252;LC_NUMERIC=C;LC_TIME=English_United Kingdom.1252
>   attached base packages:
> [1] "tools"     "stats"     "graphics"  "grDevices" "utils"     "datasets"  "methods"   "base"     
>   other attached packages:
> barley1cdf      limma       affy     affyio    Biobase 
>   "1.14.0"   "2.9.13"   "1.12.2"    "1.2.0"   "1.12.2"

I don't know that anybody will be able to help you with what you 
perceive to be a problem. Gordon Smyth has reassured you several times 
that your limma code is correct. We have been building these cdf 
packages for several years now without anybody claiming that there is a 
mapping mistake (and really, what are the odds that a mistake in the cdf 
could magically make all of your differential expression go one way - I 
have a hard time imagining how that would work).

In addition, the data and experimental conditions are your own. Instead 
of assuming that there is some mistake in the cdf, doesn't it make more 
sense to say 'Hmm, I wonder if I can confirm these results using qRT-PCR 
(or whatever method you prefer).'?

Best,

Jim



>    
>   Looking forward to hearing your comments,
>   Lev.
> 
> Lev Soinov <lev_embl1 at yahoo.co.uk> wrote:
>   Hi James, List,
> My previous e-mail was bounced because of the attachment, so i decided to send it without the one this time.
> This is related to my previous questions about barley1.cdf.
> 
> I do not have any evidence for mistakes in barley1.cdf (I do not have ANY experience in making CDFs). However, I am getting a little bit strange results second time in a row. I have e-mailed to the list some time ago about Barley arrays and paired design in LIMMA. The thing was that I was getting only upregulated genes in the expreiment. Gordon kindly helped to conclude that the results were genuine/correct. But now I am analysing a new dataset with the same amount of data (3 pairs of barley arrays, 6 arrays in total), I am using the same scripts as before (as described in the LIMMA user guide, p.40, for paired design), below. The script is very simple and is exactly as in the user guide. And once again I am getting only upregulated genes, no downregulation at all.
> This time I have a different treatment and it seems VERY strange that there is no downregulated genes at all again. Therefore I started to suspect some mistake in the cdf environment.
> 
> Thank you very much for all your help!
> Lev.
> 
> memory.limit(size = 2048)
> data<-ReadAffy()
> temp<-rma(data)
> targets <- readTargets("Targets.txt")
> Pair <- factor(targets$Pair)
> Treat <- factor(targets$Treatment, levels=c("A","B"))
> design <- model.matrix(~Pair+Treat)
> fit_pair <- lmFit(temp, design)
> fit_pair <- eBayes(fit_pair)
> topTable(fit_pair, coef="TreatB")
> 
> Targets file:
> FileName Pair Treatment
> 1.CEL 1 A
> 2.CEL 1 B
> 3.CEL 2 A
> 4.CEL 2 B
> 5.CEL 3 A
> 6.CEL 3 B
> 
> 
>>sessionInfo()
> 
> R version 2.4.1 (2006-12-18) 
> i386-pc-mingw32 
> locale:
> LC_COLLATE=English_United Kingdom.1252;LC_CTYPE=English_United Kingdom.1252;LC_MONETARY=English_United Kingdom.1252;LC_NUMERIC=C;LC_TIME=English_United Kingdom.1252
> attached base packages:
> [1] "tools" "stats" "graphics" "grDevices" "utils" "datasets" "methods" "base" 
> other attached packages:
> mouse4302cdf limma affy affyio Biobase 
> "1.5.1" "2.9.8" "1.12.2" "1.2.0" "1.12.2" 
> 
> 
> 
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-- 
James W. MacDonald
University of Michigan
Affymetrix and cDNA Microarray Core
1500 E Medical Center Drive
Ann Arbor MI 48109
734-647-5623



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