[BioC] Query regarding Bioconductor package

[Sean Davis]

Hi, Varpal.  Please keep emails going to the list so you have the best
chance of getting correct answers.

On Tue, Apr 6, 2010 at 1:12 AM, varpal singh <gilvarpal at gmail.com> wrote:
> Thanks very much for reply !!!!
>
> I want to analysis Affymetrix data by bioconductor.
> CEL files for Analysis :
> wild(GSM237292.CEL, GSM237293.CEL),treatment (GSM237294.CEL, GSM237295.CEL)
>
> I got result by running the following commands:
>
> I have a wild and a mutant types each have two replicate that you can see
> above
> In first step load
> library(affy) # load library#
> dat<-ReadAffy() #read cel file in readaffy#
> dat2<-rma(dat) #normalized data#
> dat.n<-exprs(dat2)#change in expression value#
> groups<-c("C", "C", "T1", "T1")#design a model matrix for filtering the
> data#
> groups<-as.factor(groups)
> design<-model.matrix(~groups)
> library(limma)#load limma package#
> fit<-lmFit(dat.m, design)#use linnear model#
> fit<-eBayes(fit)
> topTable(fit, coef=2)#use coefficent #
> tt<-toptable(fit, coef=2, n=nrow(dat.m))#save all expression in tt#
> rn<-rownames(tt)[tt$P.Value<-
> 0.001]# only those gene expression exist after this command which have
> P.Value <0.001#
> P.Value which are adjusted.
> I got only 137 genes,

The P.Value column is NOT adjusted for multiple testing.  The
adj.P.value column is.

> The result that you can see in the folowing attached excel files, in which
> have two sheet  in first have result of bioconductor, and other I try to
> understand how can calculate Fold change value.
> Can we predict up/down regulated genes on the basis of Fold change value
> only  ??

As I said in my last email, the log fold change will be positive for
upregulation and negative for downregulation.  The sign depends, of
course, on how you set up the analysis, but I believe that what you
have shown above will give the appropriate results.

Sean

> On Mon, Apr 5, 2010 at 10:09 PM, Sean Davis <seandavi at gmail.com> wrote:
>>
>> On Sat, Apr 3, 2010 at 1:50 AM, varpal singh <gilvarpal at gmail.com> wrote:
>> > Respected Mam/Sir,
>>
>> Hi, Varpal.
>>
>> > Myself Varpal singh from india, am doing a project on microarray data
>> > analysis by bioconductor package but am not able to interpretate
>> > results. I
>> > want to analysis Affymetrix data by bioconductor. I got result by
>> > running
>> > the following commands:
>>
>> What commands?  What is the output of sessionInfo()?  Have you read
>> the posting guide?  It will help you to get the best answers to your
>> questions.
>>
>> > I have a wild and a mutant types each have two replicate i.e Two for
>> > wild
>> > and two for mutant.
>> >
>> > I got only 137 genes. after the cut-off 0.001 for p value.
>>
>> We do not know what would be expected given the biology and we do not
>> know what analysis was done.  Also, it isn't clear what the p-value
>> represents.  Is it corrected for multiple tests?
>>
>> > The result that you can see in the folowing attached excel files, in
>> > which
>> > you can see the fold change value that i dont understand,
>>
>> File attachments are not included.
>>
>> > Can we predict up/down regulated genes on the basis of Fold change value
>> > ??
>>
>> A fold change greater than 1 means upregulation and less than 1 means
>> downregulation.  In log space, this will correspond to >0 and <0
>> respectively.
>>
>> > How is it calculate fold change between four CEL files ???
>>
>> You haven't told us enough information to give you a detailed answer,
>> but it will be something like the (average of one group)/(average of
>> the other group).
>>
>> > Is it use average of wild type replicates and mutant type replicates ??
>>
>> It sounds like you may need to get some statistics guidance, as
>> understanding the answers to the questions above are intrinsic to any
>> microarray analysis.  I would recommend seeking out such a
>> collaborator, if for no other reason than to learn a bit more about
>> microarray analysis.
>>
>> Sean
>
>