Hi,

I would like to ask everyone some questions.

I am doing a genome wide screening of siRNA.

At the end of data analysis, I manage to generate the correct data but fail to annotate the wells.

Even though the state is TRUE for annotation but they are not in html report.

State:
configured=TRUE, annotated=TRUE

x <- readPlateList("platelist.txt", name = expName, path=dataPath)
x<-configure(x,confFile = confFile, logFile = logFile,descripFile = desFile, path = dataPath)
x<-annotate(x,geneIDFile="annotationST1.txt",path=dataPath)

    xn<-summarizeChannels(x,fun = function (r1,r2,r3,r4,r5,r6,r7) r5/r3)
  

    xnp <- normalizePlates(xn, scale = "multiplicative", log = TRUE, method = "negatives", varianceAdjust = "none")
    xsc <- scoreReplicates(xnp, sign = "+", method = "zscore")
    xf <- summarizeReplicates(xsc, summary = "mean")

I tried to annotate the x (raw) and xf (summarized) one, but they generated the same results.

Plate    Well    GeneID    
1    A01    DMPK    
1    A02    empty    
1    A03    TTK    
1    A04    PIK3CA    
1    A05    SYK    
1    A06    KIAA1804    
1    A07    EPHA8    
1    A08    NAGK    
1    A09    empty    
1    A10    IRAK1    
total 384.

Please someone help me with this.

Thank you.

Hi,

I tried it.

> head(fData(xsc))
  plate well controlStatus
1     1  A01        sample
2     1  A02           neg
3     1  A03        sample
4     1  A04        sample
5     1  A05        sample
6     1  A06        sample
>
> xsc<-annotate(xsc,geneIDFile="annotationST1.txt",path=dataPath)
> head(fData(xsc))
  plate well controlStatus   GeneID  X
1     1  A01        sample     DMPK NA
2     1  A02           neg    empty NA
3     1  A03        sample      TTK NA
4     1  A04        sample   PIK3CA NA
5     1  A05        sample      SYK NA
6     1  A06        sample KIAA1804 NA

It seems like annotation function is working. But there is another column exist called "x".

At first, x was assigned as my raw data.

However, If i assigned xfw to scored, it wont work since xfw doesnt have the scored data.

With the same function, my html result doesnt show annotations to my wells,

Great, so the annotate function is working. Can you try moving the annotation step to earlier in your pipeline e.g. after configure. The annotation will get carried along. Try running

state(x)
state(xnp)
state(xf)

and so on to follow what objects have been properly annotated. Since xf comes later in the pipeline, running annotate on that will not cause the previously saved objects to also inherit the annotation. Please let me know how this affects your report.

An update to the thread for completeness. User wstoo1 sent me an example dataset privately. We figured out that he was assuming that the 'wellAnno' column should contain gene identifiers, which is not a valid expectation. The 'wellAnno' column contains information about the control status of different wells, while gene identifiers are stored in a separate column, 'GeneID'. The package is annotating the screen properly and outputting screen annotation to the report correctly.