Dear mailers,

I am trying to use your AgiMicroRna package to analyze my microarray data. I
have created the targets files (I just have one control and one test file
without any replicates). I am experiencing a problem at the filtering step
and
it shows the error "Error in is(ddNORM, "uRNAList") : object 'ddNORM' not
found".

I can not figure this out even searching for the error in different forums
and
manuals. I am attaching the codes that I have used. Please help me fix this
issue.

Thank you so much.

My code is shown below:

> setwd("C:/Documents and Settings/555/My Documents/miRNA")
> targets.micro=readTargets(infile="targets.txt",verbose=TRUE)

Target File
                                                FileName Treatment GErep
B1=miRNA_1_1=Control G2 B1=miRNA_1_1=Control G2.txt         C     1
B2=miRNA_1_2=Treated-G2           B2=miRNA_1_2=Treated-G2.txt   Treated
2

> dd.micro=readMicroRnaAFE(targets.micro,verbose=TRUE)
reading file  1  -  B1=miRNA_1_1=Control G2.txt
reading file  2  -  B2=miRNA_1_2=Treated G2.txt

 uRNAList:
       dd$TGS:         'gTotalGeneSignal'
       dd$TPS:         'gTotalProbeSignal'
       dd$meanS:       'gMeanSignal'
       dd$procS:       'gProcessedSignal'

> qcPlots(dd.micro,offset=5,
+  MeanSignal=TRUE,
+  ProcessedSignal=FALSE,
+  TotalProbeSignal=FALSE,
+  TotalGeneSignal=FALSE,
+  BGMedianSignal=FALSE,
+  BGUsed=FALSE,
+  targets.micro)
Error in plot.hclust(hc, labels = samples, main = "") :
 invalid dendrogram input
> cvArray(dd.micro,
+ "MeanSignal",
+ targets.micro,
+ verbose=TRUE)
Foreground: MeanSignal

       FILTERING BY ControlType FLAG

 RAW DATA:                       15739
 PROBES without CONTROLS:        15024
----------------------------------
 (Non-CTRL) Unique Probe:  2689
 (Non-CTRL) Unique Genes:  939
----------------------------------
DISTRIBUTION OF REPLICATED NonControl Probes
reps
  4    5    6    8   16
1512  118   59  996    4
------------------------------------------------------
Replication at Probe level- MEDIAN  CV
B1=miRNA_1_1=Control G2      B2=miRNA_1_2=Treated-G2
                    0.285                      0.270
------------------------------------------------------
DISTRIBUTION OF REPLICATED Noncontrol Genes
reps
 16
939
------------------------------------------------------
> ddTGS=tgsMicroRna(dd.micro,
+ half=TRUE,
+ makePLOT=FALSE,
+ verbose=FALSE)
> ddNORM=tgsNormalization(ddTGS,
+ "quantile",
+ makePLOTpre=TRUE,
+ makePLOTpost=TRUE,
+ targets.micro,
+ verbose=TRUE)
Error in plot.hclust(hc, labels = samples, main = "") :
 invalid dendrogram input
> ddTGS.rma=rmaMicroRna(dd.micro,
+ normalize=TRUE,
+ background=TRUE)
Calculating Expression
> MMM=ddTGS.rma$meanS
> colnames(MMM)=colnames(dd.micro$meanS)
> maintitle='TGS.rma'
> colorfill='blue'
> ddaux=ddTGS.rma
> ddaux$meanS=MMM
> mvaMicroRna(ddaux,maintitle,verbose=TRUE)

------------------------------------------------------
mvaMicroRna info:
 FEATURES :      939
 POSITIVE CTRL:          12
 NEGATIVE CTRL:          7
 STRUCTURAL:             3
> rm(ddaux)
> RleMicroRna(MMM,"RLE TGS.rma",colorfill)
> boxplotMicroRna(MMM,maintitle,colorfill)
> plotDensityMicroRna(MMM,maintitle)
> ddPROC=filterMicroRna(ddNORM,
+ dd.micro,
+ control=TRUE,
+ IsGeneDetected=TRUE,
+ wellaboveNEG=FALSE,
+ limIsGeneDetected=75,
+ limNEG=25,
+ makePLOT=FALSE,
+ targets.micro,
+ verbose=TRUE,
+ writeout=FALSE)
FILTERING PROBES BY FLAGS

Error in is(ddNORM, "uRNAList") : object 'ddNORM' not found

-- 
Karthik K.N

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