[BioC] Limma_Agilent_2color_Array

[Wolfgang Huber]

Dear Abhilash,

 > The array1is a NChannelSet created from the RGList using the following
 > command.
 > array1<-new("NChannelSet")

Why do you say that array1 was "from the RGList", since with this line 
you are creating an empty NChannelSet, with no data in it. Hence the 
(admittedly not very descriptive, but totally correct) error message.

Please consider using the "as" method from the "convert" package to 
convert an RGList into an NChannelSet. For a more explicit description, 
see also 
http://www.bioconductor.org/packages/2.1/data/experiment/vignettes/CCl4/inst/doc/CCl4.pdf

  Best wishes
	Wolfgang

> The following is the output of sessionInfo().
> R version 2.6.1 (2007-11-26)
> i386-pc-mingw32
> 
> locale:
> LC_COLLATE=English_United States.1252;LC_CTYPE=English_United
> States.1252;LC_MONETARY=English_United
> States.1252;LC_NUMERIC=C;LC_TIME=English_United States.1252
> 
> attached base packages:
>  [1] grid      splines   tools     stats     graphics  grDevices utils
> datasets  methods   base
> 
> other attached packages:
>  [1] arrayQualityMetrics_1.2.1 latticeExtra_0.3-1
> CCl4_1.0.7                simpleaffy_2.14.05
>  [5] affyPLM_1.14.0            gcrma_2.10.0
> matchprobes_1.10.0        RColorBrewer_1.0-2
>  [9] vsn_3.2.1                 limma_2.12.0
> affy_1.16.0               preprocessCore_1.0.0
> [13] affyio_1.6.1              genefilter_1.16.0
> survival_2.34             geneplotter_1.16.0
> [17] lattice_0.17-2            annotate_1.16.1
> xtable_1.5-2              AnnotationDbi_1.0.6
> [21] RSQLite_0.6-8             DBI_0.2-4
> Biobase_1.16.3
> 
> loaded via a namespace (and not attached):
> [1] KernSmooth_2.22-21
> 
> The array1is a NChannelSet created from the RGList using the following
> command.
> array1<-new("NChannelSet")
> 
> Regards,
> Abhilash
> 
> On Fri, Apr 18, 2008 at 8:04 PM, Wolfgang Huber <huber at ebi.ac.uk> wrote:
> 
>> Dear Abhilash
>>
>>> As you directed, I tried arrayQualityMetrics. I used feature extracted
>> txt
>>> file  from Agilent  arrays and created RGList. It has given the
>> following
>>> error, I am wondering whether it is  because I didnot try to create
>>> assayData, featureData and phenoData.
>>> arrayQualityMetrics(expressionset = array1,
>>> + outdir = "array")
>>> *The directory 'array' has been created.
>>> Error in seq_len(ncol(x)) : argument must be non-negative
>>>
>>> *What could I do to solve this problem?
>> First of all, give the output of sessionInfo().
>> Second, you could tell us a bit more about what "array1" is.
>>
>>  Best wishes
>>         Wolfgang
>>
>>> Regards,
>>> Abhilash
>>>
>>>
>>> On Wed, Apr 16, 2008 at 11:07 PM, Wolfgang Huber <huber at ebi.ac.uk>
>> wrote:
>>>> Dear Abilash,
>>>>
>>>> you could try to run the arrayQualityMetrics report from the eponymous
>>>> package (and preferably a current devel version [1]). The plots in
>> there
>>>> would help you to assess both whether there are gross problems in the
>>>> data, and how much and what type of normalisation is needed.
>>>>
>>>> Adequate normalisation removes unwanted technical variation while it
>>>> maintains the interesting biological signal in the data. The best way
>> to
>>>> assess whether this is goal is achieved is by looking at the behaviour
>>>> of the controls that were done as part of the experiment.
>>>>
>>>> [1]
>>>>
>>>>
>> http://www.bioconductor.org/packages/2.2/bioc/html/arrayQualityMetrics.html
>>>> Best wishes
>>>>  Wolfgang
>>>>
>>>> ------------------------------------------------------------------
>>>> Wolfgang Huber  EBI/EMBL  Cambridge UK  http://www.ebi.ac.uk/huber
>>>>
>>>>
>>>> 16/04/2008 18:02 Abhilash Venu a écrit
>>>>> Hi all,
>>>>>
>>>>> I have a few questions. I am analyzing  20 experiments  performed  in
>>>>> Agilent 4x44K. Tumor and adjacent normal were labeled with Cy5 and Cy3
>>>>> respectively. The experiments include two dye swaps also. The feature
>>>>> extraction were performed using Agilent feature extraction software
>>>> version
>>>>> 8.5. The txt files obtained after feature extraction, subjected to
>>>> further
>>>>> analysis by limma. The script has given below
>>>>> txt_files <- dir(pattern=".txt")
>>>>> RG<-read.maimages(txt_files, source="agilent")
>>>>> design<-c(1,1,1,1,1,1,1,1,1,1,1,-1,1,1,1,1,1,1,1,-1) # this include
>> two
>>>> dye
>>>>> swap also
>>>>> Rgene<-backgroundCorrect(RG,method="normexp")
>>>>> fit<-lmFit(Rgene,design)
>>>>> fit<-eBayes(fit)
>>>>> topTable(fit,adjust="fdr", number=100)
>>>>>
>>>>> As a result I get the format in which 'B' indicates what parameter?
>> And
>>>> is
>>>>> this analysis method is enough to get a good data?
>>>>> In Agilent arrays, I think generally it is normalized, in that case
>> how
>>>> the
>>>>> data will be affected by normalizing it again using above method?
>>>>> How can we decide which normalization methods should be used? Do you
>>>> think
>>>>> its always better to decide by plotting different graphs?
>>>>>
>>>>> Comments and answers will be appreciated.
>>>>>
>>>>> Regards,
>>>>> Abhilash
>>>>>
>>>>>
>>>
>>>
> 
> 
> 

-- 
Best wishes
  Wolfgang

------------------------------------------------------------------
Wolfgang Huber  EBI/EMBL  Cambridge UK  http://www.ebi.ac.uk/huber