[BioC] making a cdf package for a custom affymetrix array and set up for limma
James W. MacDonald
jmacdon at uw.edu
Thu Nov 8 14:51:49 CET 2012
Hi Lauren,
On 11/7/2012 8:04 PM, Lauren Sassoubre wrote:
> Thanks Jim!
>
> I realized that the code was calling on a cdf file with a typo in the
> name. After I corrected the cdf file name, make.cdf.package worked and
> I was able to install it in the terminal window.
>
> I would like to use limma to analyze the data. I have a treatment
> (light) and a control (dark), 5 time points for each (i'm considering
> lights and darks paired for each time point), and 3 sets of
> experiments (biological replicates) so I have a total of 30 arrays. I
> made a txt file (see below) with information on the filename and
> corresponding treatment and experiment. *I'm not sure how to include
> the information about which are paired within this file??*
>
> The data has been loaded with ReadAffy and processed with the rma
> function:
> cel <- ReadAffy()
> pre<-rma(cel)
> eset <- exprs(pre)
This last step is unnecessary. The vast majority of functions in BioC
are designed to work on ExpressionSet objects, so there is no need to
extract the expression data. In other words, you will get the same
results if you do
fit <- lmFit(pre, design)
or
fit <- lmFit(eset, design)
And the upside of using an ExpressionSet is you a) get to append
phenotypic and other data with the object, and b) you can manipulate the
ExpressionSet as if it were a matrix, and everything gets subsetted
accordingly. So
pre[,1:3]
will do exactly what you would expect, resulting in an ExpressionSet
containing data for only the first three samples.
>
> *How do I combine the file with the information about filename,
> treatment and experiment (see below) with my processed data using
> factor and model matrix??*
> I'm trying to use/understand the code in section 8.8 of the Limma user
> guide:
I assume that is a typo and you meant section 8.6. But I wonder why you
are emulating that section, given it shows how to analyze a timecourse
experiment, and it appears your experiment isn't one?
I think you would be better off looking at section 8.4.1, which shows
how to do a paired experiment by blocking on subject. The file you show
below doesn't contain the pairing information, but what you want to do
is add a column similar to the SibShip column in 8.4.1 that you can use
as a blocking variable.
Best,
Jim
>
> lev <- c("wt.0hr","wt.6hr","wt.24hr","mu.0hr","mu.6hr","mu.24hr")
> f <- factor(targets$Target, levels=lev)
> design <- model.matrix(~0+f)
> colnames(design) <- lev
> fit <- lmFit(eset, design)
>
> This is what the txt file with information on filename, treatment, and
> experiment looks like:
>
> Filename Treatment experiment
> EF731DO.CEL dark 1
> EF731D12.CEL dark 1
> EF731D2.CEL dark 1
> EF731D24.CEL dark 1
> EF731D6.CEL dark 1
> EF731LO.CEL light 1
> EF731L12.CEL light 1
> EF731L2.CEL light 1
> EF731L24.CEL light 1
> EF731L6.CEL light 1
> EF813DO.CEL dark 2
> EF813D12.CEL dark 2
> EF813D2.CEL dark 2
> EF813D24.CEL dark 2
> EF813D6.CEL dark 2
> EF813LO.CEL light 2
> EF813L12.CEL light 2
> EF813L2.CEL light 2
> EF813L24.CEL light 2
> EF813L6.CEL light 2
> EF815DO.CEL dark 3
> EF815D12.CEL dark 3
> EF815D2.CEL dark 3
> EF815D24.CEL dark 3
> EF815D6.CEL dark 3
> EF815LO.CEL light 3
> EF815L12.CEL light 3
> EF815L2.CEL light 3
> EF815L24.CEL light 3
> EF815L6.CEL light 3
>
>
> Thanks in advance for your help!!
> Best,
> Lauren
> (graduate student)
>
>
> On Nov 7, 2012, at 12:27 PM, James W. MacDonald wrote:
>
>> Hi Lauren,
>>
>> Please don't take conversations off-line; we hope that the archived
>> questions and answers will be useful for others.
>>
>> On 11/7/2012 3:10 PM, Lauren Sassoubre wrote:
>>> Thank you Jim!
>>>
>>> If you don't mind me asking another question, I was able to make a
>>> cdf env last week but deleted it and now when I try to use the
>>> function I get the error message I posted. I'm not sure what
>>> changed. Are previously created cdf environments saved in some way
>>> that would complicate making another with the same cdf file?
>>
>> You could have saved the old environment if when you quit R you said
>> 'yes'. It will automatically be dumped into a file .RData, which will
>> automatically be loaded if you start R in that same directory. If
>> this is true, you will get a message
>>
>> [Previously saved workspace restored]
>>
>> after you start R. In which case you can do ls() to see what is in
>> the saved workspace, and maybe your old env is there.
>>
>> However, this shouldn't interfere with the creation of a new
>> environment. You can always overwrite things in the .GlobalEnv (the
>> default workspace).
>>
>> You can always see if the file is where you think it is. Start R, and
>> then do a
>>
>> dir(".", pattern = "[Cc][Dd][Ff]")
>>
>> if it responds with a character(0), then the cdf isn't in your
>> working directory. You then have to use setwd() to change to the
>> correct dir, or just point to the directory where you have the cdf.
>>
>> Best,
>>
>> Jim
>>
>>
>>> Thanks again!
>>> Lauren
>>>
>>>
>>> On Nov 7, 2012, at 7:20 AM, James W. MacDonald wrote:
>>>
>>>> Hi Lauren,
>>>>
>>>> On 11/6/2012 9:11 PM, Lauren Sassoubre wrote:
>>>>> Hi,
>>>>> This error message has come up in previous posts but I haven't
>>>>> found a solution that works. I'm having trouble loading a cdf file
>>>>> for a custom array and using that cdf for the function ReadAffy.
>>>>> Below are the code and error message. What does this error mean? I
>>>>> have tried reinstalling R and deleting the cdf files and reloading
>>>>> them from the original disk. I'm working with the most recent
>>>>> version of R for Macs.
>>>>> Thanks in advance for the help!
>>>>> Lauren
>>>>>
>>>>>> make.cdf.env("Gilmorea520817.cdf")
>>>>> Error in isCDFXDA(file.path(path.expand(cdf.path), filename)) :
>>>>> Unable to open the file
>>>>> /Users/lmsassoubre/Desktop/solarsimandmicroarrays/CD_Gilmorea520187F/Full/Gilmorea520817.cdf
>>>> There are two obvious possibilities. First, the file might not be
>>>> in the path you specify. For example:
>>>>
>>>>> dir(".", "nopackage.cdf")
>>>> character(0)
>>>>> make.cdf.env("nopackage.cdf")
>>>> Error in isCDFXDA(file.path(path.expand(cdf.path), filename)) :
>>>> Unable to open the file /misc/staff/jmacdon/nopackage.cdf
>>>>
>>>> alternatively, you might not have read permissions. This is much
>>>> less likely, especially if you are copying the file and running R
>>>> as the same user. To make the below work, I had to change
>>>> permissions to a state that is unlikely to exist in that situation.
>>>> However unlikely, it is still a possibility:
>>>>
>>>>> system("ls -lah AG.CDF")
>>>> --wx------ 1 jmacdon staff 20M Nov 7 07:13 AG.CDF
>>>>> make.cdf.env("AG.CDF")
>>>> Error in isCDFXDA(file.path(path.expand(cdf.path), filename)) :
>>>> Unable to open the file /misc/staff/jmacdon/AG.CDF
>>>>
>>>> I suppose there are other alternatives as well, but none come
>>>> readily to mind.
>>>>
>>>> Best,
>>>>
>>>> Jim
>>>>
>>>>
>>>>> _______________________________________________
>>>>> Bioconductor mailing list
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>>>> --
>>>> James W. MacDonald, M.S.
>>>> Biostatistician
>>>> University of Washington
>>>> Environmental and Occupational Health Sciences
>>>> 4225 Roosevelt Way NE, # 100
>>>> Seattle WA 98105-6099
>>>>
>>
>> --
>> James W. MacDonald, M.S.
>> Biostatistician
>> University of Washington
>> Environmental and Occupational Health Sciences
>> 4225 Roosevelt Way NE, # 100
>> Seattle WA 98105-6099
>>
>
--
James W. MacDonald, M.S.
Biostatistician
University of Washington
Environmental and Occupational Health Sciences
4225 Roosevelt Way NE, # 100
Seattle WA 98105-6099
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