[BioC] problems about cDNA vs genomic arrays normalization

[yanju]

Hello Jenny,

I also realized the first problem you mentioned after I sent the email 
to you. After I change it, the results seems better, not very much 
markers appeared in the list. Thank you very much for your suggestion 
during these two days. I think for the time being, my questions have 
been settled.

Have a nice day,
Yanju
Jenny Drnevich wrote:

> Hi Yanju,
>
> Two suggestions - 1) The code I gave you before was written as if your 
> reference was in the green (Cy3) channel. However, based on the 
> results of your 'modelMatrix(targets, ref="gDNA")' command, your 
> reference is in the red (Cy5) channel. Therefore, you would have to 
> reverse some of the commands where appropriate (e.g., use method 
> "Rquantile" and replace M values with G values).
>
> 2) Find a local statistician to consult about the analysis, because it 
> appears you have a 2 x 7 factorial design (2 strains and 7 timepoints 
> = 14 treatment groups total). There are variety of ways to analyze 
> this experimental design, depending on what all you want to know from 
> the data. If you really only wanted to know which genes were different 
> between mu & wt at each time point independently, then you could 
> analyze  the arrays from each time point separately. However, there is 
> so much more information to be gained from this data set, which is why 
> I suggest you consult a local statistician.
>
> Best of luck,
> Jenny
>
> At 10:54 AM 11/21/2006, yanju wrote:
>
>> Hello Jenny,
>>
>> I adapted my code according to your suggestion. Then at some time 
>> points, the results showed that the most differently expressed genes 
>> are markers. This is every werld.
>>
>> And It doesnt matter if I change -1 in the design matrix to 1 (my 
>> method: new design matrix=old design matrix* -1, old design matrix 
>> was derived from modelMatrix function) or not. I mean this didnt 
>> effect my results.
>>
>> Since I could not figure it out, I paste my code here. Hope you could 
>> tell me what's wrong with my program. Basic information of the data:
>> Two-channle array, 7 time points from 16-72h, at each time point 
>> there are some repelicants. Aim is to detect the different expressed 
>> genes at each time points.
>>
>> From the very begin of the code:
>>
>> targets<-readTargets("target_new_16_72.txt")
>> rg<-read.maimages(targets, source="genepix",wt.fun=wtflags(0.1))
>>       # read targets and genepix files
>>
>> rgc<-backgroundCorrect(rg, method="half")
>>       # bacground correction
>>
>> MA.Gquant<-normalizeBetweenArrays(rgc, method="Gquantile")
>> RG.Gquant<-RG.MA(MA.Gquant)
>> MA.fake<-MA.Gquant
>> MA.fake$M<-log2(RG.Gquant$R)
>>       #normalization
>>
>> design<-modelMatrix(targets, ref="gDNA")
>> design_revise<-design*-1
>>       #design was similar like follows.
>>       #   wt16 wt20 wt24
>>       #[1,]    -1    0    0
>>       #[2,]     0    -1    0
>>       #[3,]     0    0    -1
>>       #Then it was multiply by -1 to  have the positive value.
>>
>>
>>    fit<-lmFit(MA.fake,design_revise)
>>    cont.matrix<-makeContrasts(MUvsWT16=mu16-wt16, MUvsWT20=mu20-wt20, 
>> MUvsWT24=mu24-wt24, MUvsWT36=mu36-wt36, MUvsWT48=mu48-wt48,
>>    MUvsWT60=mu60-wt60, MUvsWT72=mu72-wt72,levels=design_revise)
>>    fit2<-contrasts.fit(fit,cont.matrix)
>>    fit2<-eBayes(fit2)
>>       #fit the data to linear model and Bayes statistical summary
>>
>> result20<-topTable(fit2,coef=2, number=20,adjust="BH")
>>       #detecting the top20 differently expressed genes at time point20.
>>       #But as I said, most of the top20 were markers or the "no spot"
>>
>> Hope you could help me figure out the problems. I really appreciate 
>> your help. Thanks.
>>
>> Regards,
>> Yanju
>>
>>
>
> Jenny Drnevich, Ph.D.
>
> Functional Genomics Bioinformatics Specialist
> W.M. Keck Center for Comparative and Functional Genomics
> Roy J. Carver Biotechnology Center
> University of Illinois, Urbana-Champaign
>
> 330 ERML
> 1201 W. Gregory Dr.
> Urbana, IL 61801
> USA
>
> ph: 217-244-7355
> fax: 217-265-5066
> e-mail: drnevich at uiuc.edu