Hello,

I am making progress in learning R but I must admit that I am really slow and without all your help I would have given up on this. I still have some recurring troubles with Agi4x44PreProcess. This time I am having issues with Replicated genes (genes.rpt.agi). It looks like I am missing something. I have posted the session info here and highlighted the problematic ones in "red". I really appreciate your help.

Thank you very much in advance,

Sincerely, Neel

[R.app GUI 1.29 (5464) i386-apple-darwin8.11.1]

> source("http://bioconductor.org/biocLite.R")
> biocLite()
Using R version 2.9.2, biocinstall version 2.4.13.
Installing Bioconductor version 2.4 packages:
 [1] "affy"         "affydata"     "affyPLM"      "annaffy"      "annotate"     "Biobase"      "biomaRt"     
 [8] "Biostrings"   "DynDoc"       "gcrma"        "genefilter"   "geneplotter"  "hgu95av2.db"  "limma"       
[15] "marray"       "multtest"     "vsn"          "xtable"       "affyQCReport"
Please wait...
> library(org.Dr.eg.db)
Loading required package: AnnotationDbi
Loading required package: Biobase
> setwd("/Users/Neel/agilent")
> getwd()
[1] "/Users/Neel/agilent"
> library("Agi4x44PreProcess")
Loading required package: limma
Loading required package: annotate
Loading required package: genefilter
> targets=read.targets(infile="infile.txt")
 
Target File 
          X  FileName Treatment GErep
conta cont1 conta.txt   control     1
contb cont2 contb.txt   control     2
contc cont3 contc.txt   control     3
contd cont4 contd.txt   control     4
pcba   pcb1  pcba.txt       pcb     1
pcbb   pcb2  pcbb.txt       pcb     2
pcbc   pcb3  pcbc.txt       pcb     3
pcbd   pcb4  pcbd.txt       pcb     4
 
> aa=read.AgilentFE(targets, makePLOT=FALSE)
Read conta.txt 
Read contb.txt 
Read contc.txt 
Read contd.txt 
Read pcba.txt 
Read pcbb.txt 
Read pcbc.txt 
Read pcbd.txt 
 
  RGList: 
	dd$R:	'gProcessedSignal'  
	dd$G:	'gMeanSignal'  
	dd$Rb:	'gBGMedianSignal'  
	dd$Gb:	'gBGUsed'  
 
> aaNORM = BGandNorm(aa, BGmethod = "half", NORMmethod = "quantile", foreground = "MeanSignal", background = "BGMedianSignal", offset = 50, makePLOTpre = FALSE, makePLOTpost = FALSE)
Loading required package: vsn
BACKGROUND CORRECTION AND NORMALIZATION  

	foreground: MeanSignal 
	background: BGMedianSignal 

	BGmethod:	 half 
	NORMmethod:	 quantile 
	OUTPUT in log-2 scale 
> CV.rep.probes(aa, "org.Dr.eg.db", foreground="MeanSignal", raw.data= TRUE, writeR=FALSE,targets)

------------------------------------------------------ 
Non-CTRL Replicated probes 
	foreground:  MeanSignal 
		FILTERING BY ControlType FLAG 
		RAW DATA: PROBES AFTER ControlType FILTERING:  42990 

------------------------------------------------------ 
	REPLICATED NonCtrl Probes 21495 
	UNIQUE probes 21495 
	DISTRIBUTION OF REPLICATED NonControl Probes 
reps
    1 
21495 
	# REPLICATED (redundant) probeNames 21495 
------------------------------------------------------ 
MEDIAN % CV 
conta contb contc contd  pcba  pcbb  pcbc  pcbd 
2.378 0.963 1.233 1.997 2.439 1.282 1.438 2.104 

> genes.rpt.agi(aa, "org.Dr.eg", raw.data = TRUE, WRITE.html = FALSE, REPORT = FALSE)

GENE SETS: same genes interrogated by different probes 
		FILTERING BY ControlType FLAG 
		RAW DATA: PROBES AFTER ControlType FILTERING:  42990 

	INPUT DATA: RAW 
	CHIP: org.Dr.eg 

	PROBE SETS (NON-CTRL prob rep. x 10):	 21495 
Error in lookUp(PROBE_ID, annotation.package, "SYMBOL") : 
  No keys provided  (Can anyone explain to me what keys means in R?)


> PROBE_ID = aa$ProbeUID$ProbeName
> GENE_ID = unlist(lookUp(PROBE_ID, "org.Dr.eg.db", "org.Dr.egACCNUM") )

Error in lookUp(PROBE_ID, "org.Dr.eg.db", "org.Dr.egACCNUM") : 
  No keys provided

> head <- c("PROBE ID","org.Dr.egACCNUM","SYMBOL")
> ensembl.htmlpage(PROBE_ID,filename,"org.Dr.eg", title, table.head=head,table.center = TRUE)

Error in match.arg(annotation.package, c("hgug4112a.db", "mgug4122a.db",  :
  'arg' should be one of “hgug4112a.db”, “mgug4122a.db”, “notAnnPack”

> ensembl.htmlpage(PROBE_ID,filename,"org.Dr.eg.db", title, table.head=head,table.center = TRUE)

Error in file(filename, "w") : cannot open the connection
In addition: Warning message:
In file(filename, "w") : cannot open file 'org.Dr.eg.db': Is a directory

(Do you think I should create annotation package to solve this?)





Neel Aluru
Postdoctoral Scholar
Biology Department
Woods Hole Oceanographic Institution
Woods Hole, MA 02543
USA
508-289-3607




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