Dear Bioc list,

I am dealing with Agilent 2-color data, generated by GenePix Pro  
6.1.0.2 with a GenePix 4000B scanner.

I am able to input and analyze them using Limma, but I am puzzled by  
Spot Types.

In the raw data files in which information for each spot is listed in  
columns, there is a column labeled as "ControlType". There are four  
values: ignore, neg, pos, and FALSE.

Obviously, all the real probes are labeled as "FALSE", and "ignore"  
was assigned to blank spot since their IDs were "NA" and their Names  
were left blank. Spots labeled "neg" or "pos" were various controls  
with different kinds of Name and ID.

 From a old post from Dr. Gordon Smyth, I can use the annotation  
option in read.maimages to input this column, by using:

 > Test<-read.maimages(targetsL$FileName, source="genepix",  
wt.fun=wtflags(weight=0, cutoff=-50), annotation=c("Block", "Row",  
"Column", "ID","Name","ControlType"))

The genes object would contain this info such as:

 > Test$genes[1:5,]
   Block Row Column              ID            Name ControlType
1     1   1      1 GE_BrightCorner GE_BrightCorner         pos
2     1   1      2 GE_BrightCorner GE_BrightCorner         pos
3     1   1      3      DarkCorner      DarkCorner         pos
4     1   1      4      DarkCorner      DarkCorner         pos
5     1   1      5      DarkCorner      DarkCorner         pos

Now the questions:

1) So I need to do the step Gordon suggested, for using the Spot  
Types info in further analysis?

2) Should I consider those BrightCorner and DarkCorner spots as  
control spots? Or I should ignore them?

3) How to deal with empty value in a Spot Types file?

In my data, those spots labeled with "ignore" have ID= "NA", and  
nothing under Name.

An example of such spot:

 > Test$genes[30,]
    Block Row Column   ID Name ControlType Status
30     1   1     30 <NA>           ignore

Note there is nothing under Name for this spot.

In my Spot Type file, I tried to use nothing under Name to let  
controlStatus to identify such spots, but it didn't seem to work:

 > spottypes
        SpotType ID            Name  Color
1          Gene  *            gb_*  black
2      PosCtrl1  *           DCP_*   blue
3      PosCtrl2  *             RC*   blue
4      PosCtrl3  *             EQC   blue
5      PosCtrl4  *            ETG*   blue
6      PosCtrl5  *           E1A_*    red
7       NegCtrl  * NegativeControl yellow
8  BrightCorner  * GE_BrightCorner  brown
9    DarkCorner  *      DarkCorner  brown
10        Blank  *                 orange

 > Test$genes$Status <- controlStatus(spottypes, Test)
Matching patterns for: ID Name
Found 42212 Gene
Found 36 PosCtrl1
Found 120 PosCtrl2
Found 40 PosCtrl3
Found 130 PosCtrl4
Found 320 PosCtrl5
Found 153 NegCtrl
Found 14 BrightCorner
Found 604 DarkCorner
Found 0 Blank
Setting attributes: values Color

I tried to use "NA" under ID, it didn't work either. Any suggestions?

In Section 4.8 of Limma User Guide, the example has values of "Blank"  
and "BLANK", do they have general meanings in Limma or R? My guess is  
they just happened to be in that example data.

Thanks very much for any inputs

Yong Yin, Ph.D.

Senior Scientist
Genome Sequencing Center
Washington University School of Medicine, Campus box 8501
4444 Forest Park
Saint Louis, MO 63108

Tel: (314) 286-1415



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