The support.bioconductor.org editor has been updated to markdown! Please see more info at: Tutorial: Updated Support Site Editor

Question: Limma import .gpr file error
0
gravatar for Guillaume Robert
4 weeks ago by
France/Nantes/Inovarion
Guillaume Robert0 wrote:

Hi all,

Sorry for this maybe naive question, but I never analysed microarray before.

I'm trying to import .gpr files from a public dataset (https://www.ebi.ac.uk/arrayexpress/experiments/E-TABM-104/) in limma thanks to the function read.maimages().

As specified in the documentation I've created a "target" file with the file names and the RNA information on cy3 and cy3, and implemented a function to filter flags, then I try to read the .gpr files that I've downloaded :

targets=readTargets("targets.csv",sep="\t", row.names="Name")

f <- function(x) as.numeric(x$Flags > -49)

RG <- read.maimages(files=targets$FileName,source="genepix",columns=list(R="GenePix:F635 Mean",G="GenePix:F532 Mean",Rb="GenePix:B635 Median",Gb="GenePix:B532 Median"), wt.fun=f)

Unfortunately I get the following error message :

Error in readGPRHeader(fullname) : 
  File is not in Axon Text File (ATF) format

After some research I think it comes from the fact that the .gpr files have no headers. But I'm not really sure if it comes from that, and if it is the case, what "standard" header I could put to have the files imported.

If anyone has encountered the same issue and would advise me it would be really helpful.

Thanks,

microarray limma gpr • 110 views
ADD COMMENTlink modified 4 weeks ago by Gordon Smyth36k • written 4 weeks ago by Guillaume Robert0
Answer: Limma import .gpr file error
4
gravatar for James W. MacDonald
4 weeks ago by
United States
James W. MacDonald49k wrote:

Here are the headers:

> scan("25A_12477146.gpr", nlines = 1, what = "c", sep = "\t")
Read 54 items
 [1] " metaColumn"                        "metaRow"                           
 [3] "column"                             "row"                               
 [5] "Reporter identifier"                "GenePix:% > B532+1SD"              
 [7] "GenePix:% > B532+2SD"               "GenePix:% > B635+1SD"              
 [9] "GenePix:% > B635+2SD"               "GenePix:Autoflag"                  
[11] "GenePix:B Pixels"                   "GenePix:B532"                      
[13] "GenePix:B532 CV"                    "GenePix:B532 Mean"                 
[15] "GenePix:B532 Median"                "GenePix:B532 SD"                   
[17] "GenePix:B635"                       "GenePix:B635 CV"                   
[19] "GenePix:B635 Mean"                  "GenePix:B635 Median"               
[21] "GenePix:B635 SD"                    "GenePix:Circularity"               
[23] "GenePix:Dia."                       "GenePix:F Pixels"                  
[25] "GenePix:F532 % Sat."                "GenePix:F532 CV"                   
[27] "GenePix:F532 Mean"                  "GenePix:F532 Mean - B532"          
[29] "GenePix:F532 Median"                "GenePix:F532 Median - B532"        
[31] "GenePix:F532 SD"                    "GenePix:F532 Total Intensity"      
[33] "GenePix:F635 % Sat."                "GenePix:F635 CV"                   
[35] "GenePix:F635 Mean"                  "GenePix:F635 Mean - B635"          
[37] "GenePix:F635 Median"                "GenePix:F635 Median - B635"        
[39] "GenePix:F635 SD"                    "GenePix:F635 Total Intensity"      
[41] "GenePix:Flags"                      "GenePix:Log Ratio (635/532)"       
[43] "GenePix:Mean of Ratios (635/532)"   "GenePix:Median of Ratios (635/532)"
[45] "GenePix:Normalize"                  "GenePix:Ratio of Means (635/532)"  
[47] "GenePix:Ratio of Medians (635/532)" "GenePix:Ratios SD (635/532)"       
[49] "GenePix:Rgn R? (635/532)"           "GenePix:Rgn Ratio (635/532)"       
[51] "GenePix:SNR 532"                    "GenePix:SNR 635"                   
[53] "GenePix:Sum of Means (635/532)"     "GenePix:Sum of Medians (635/532)"

Which are not standard, so far as I know. So you have to specify:

> columns <- list(G = "GenePix:F532 Median", Gb = "GenePix:B532 Median" ,R = "GenePix:F635 Median" , Rb = "GenePix:B635 Median")
> z <- read.maimages("25A_12477146.gpr", columns = columns)
Read 25A_12477146.gpr 
>
ADD COMMENTlink written 4 weeks ago by James W. MacDonald49k

Ok thank you very much it's working now.

ADD REPLYlink written 29 days ago by Guillaume Robert0
Answer: Limma import .gpr file error
2
gravatar for Gordon Smyth
4 weeks ago by
Gordon Smyth36k
Walter and Eliza Hall Institute of Medical Research, Melbourne, Australia
Gordon Smyth36k wrote:

To reinforce James MacDonald's answer, the raw data files for this experiment on ArrayExpress have been post-processed since being output by GenePix and are no longer in GenePix format. Hence you can't specify source="genepix". Instead you need

cols <- list(R  = "GenePix:F635 Mean,    G  = "GenePix:F532 Mean",
             Rb = "GenePix:B635 Median", Gb = "GenePix:B532 Median")
RG <- read.maimages(targets, annotation="Reporter identifier", columns=cols)

Personally I would skip the wt.fun argument, but it's up to you. The most common flags just mean that a spot has low itensity, but limma is perfectly able to deal with low intensity spots.

ADD COMMENTlink modified 28 days ago • written 4 weeks ago by Gordon Smyth36k

Thank you for your response. Why would you skipped the flag filtering? I've looked into the file and found several probes flagged with either -100, -75 or -50, but I haven't found the meaning of those flags.

ADD REPLYlink modified 29 days ago • written 29 days ago by Guillaume Robert0
1

Why would you use flag filtering if you don't know what the flags mean?

You shouldn't discard data unless there is strong reason to do so. These flags were designed by GenePix to protect some very simple types of analyses. limma however does a very robust analysis and usually doesn't need this sort of protection. I used to use the flags occasionally but gradually found it wasn't very important.

ADD REPLYlink modified 28 days ago • written 29 days ago by Gordon Smyth36k

Ok I will keep the flagged lines then. Thanks for the informations.

ADD REPLYlink written 29 days ago by Guillaume Robert0
Please log in to add an answer.

Help
Access

Use of this site constitutes acceptance of our User Agreement and Privacy Policy.
Powered by Biostar version 16.09
Traffic: 386 users visited in the last hour