Question: Failed to annotated my wells
0
gravatar for wstoo1
4.2 years ago by
wstoo10
European Union
wstoo10 wrote:

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.

 

 

cellhts2 • 621 views
ADD COMMENTlink modified 4.1 years ago by Joseph Barry160 • written 4.2 years ago by wstoo10
Answer: Failed to annotated my wells
0
gravatar for Joseph Barry
4.2 years ago by
Joseph Barry160
Dana-Farber Cancer Institute, Boston, USA
Joseph Barry160 wrote:
Hi, Can you print the feature data before and after the annotate step to clarify whether or not the annotate function did its job? e.g. the equivalent to > x <- readPlateList("Platelist.txt", path=datadir, name="KcViabSmall") KcViabSmall: found data in 16 x 24 (384 well) format. Read 6 plates. > head(fData(x)) plate well controlStatus 1 1 A01 unknown 2 1 A02 unknown 3 1 A03 unknown 4 1 A04 unknown 5 1 A05 unknown 6 1 A06 unknown > x <- annotate(x, "GeneIDs_Dm_HFAsubset_1.1.txt", path=datadir) > head(fData(x)) plate well controlStatus HFAID GeneID 1 1 A01 unknown <na> <na> 2 1 A02 unknown <na> <na> 3 1 A03 unknown HFA00274 CG11371 4 1 A04 unknown HFA00646 CG31671 5 1 A05 unknown HFA00307 CG11376 6 1 A06 unknown HFA00324 CG11723 If the annotate step worked you should see an extra column. What you expect to see in the report is potentially a separate issue. Joe On Fri, Jul 24, 2015 at 11:00 AM, wstoo1 [bioc] <noreply@bioconductor.org> wrote: > Activity on a post you are following on support.bioconductor.org > > User wstoo1 <https: support.bioconductor.org="" u="" 8468=""/> wrote Question: > Failed to annotated my wells <https: support.bioconductor.org="" p="" 70344=""/>: > > 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. > > > > > > ------------------------------ > > Post tags: cellhts2 > > You may reply via email or visit Failed to annotated my wells >
ADD COMMENTlink written 4.2 years ago by Joseph Barry160

Hi,

The original script that my supervisor sent me was below:

x <- readPlateList("platelist.txt", name = expName, path=dataPath)
    x<-configure(x,confFile = confFile, logFile = logFile,descripFile = desFile, path = dataPath)
    #Calculate the survival ratio by normalizing FL channel to the RL

    xn<-summarizeChannels(x,fun = function (r1,r2,r3,r4,r5,r6,r7) r5/r3)
    
#Normalise plates to the median of the plate values

    xnp <- normalizePlates(xn, scale = "multiplicative", log = TRUE, method = "negatives", varianceAdjust = "none")

#Score wells within each replicate by caluclating a z score for each well based on replicate median and mad
    xsc <- scoreReplicates(xnp, sign = "+", method = "zscore")

#summarize the replicates by taking the mean value for each
    xf <- summarizeReplicates(xsc, summary = "mean")

    xfw<-annotate(xf,geneIDFile="annotationST1.txt",path=dataPath)

He annotated to xfw variable, which has scores.

In the write report function, the scored input he used was xf.

out <- writeReport(raw=x, normalized=xnp, scored=xf, force = TRUE, plotPlateArgs = TRUE, imageScreenArgs = list(zrange=c( -4, 8),ar=1), map=TRUE, outdir=outPath)

Hence, the Annotated status was FALSE.

However, no matter the annotated status in write report feedback is TRUE or FALSE, the html report generated still no annotations to the wells.

HELP !!!!

ADD REPLYlink written 4.2 years ago by wstoo10
Unfortunately you just sharing the script your supervisor sent you does not give me the information I need to ascertain whether or not the annotate function worked. Please wrap the annotate command with the following 'fData' command, as I suggested in my previous post. head(fData(xf)) xfw<-annotate(xf,geneIDFile="annotationST1.txt",path=dataPath) head(fData(xfw)) Otherwise how can we know if the annotate function did its job? Also, you are using "xf" for scored in the writeReport function, but saving the annotated object to "xfw". Therefore you might also want to try out <- writeReport(raw=x, normalized=xnp, scored=xfw, force = TRUE, plotPlateArgs = TRUE, imageScreenArgs = list(zrange=c( -4, 8),ar=1), map=TRUE, outdir=outPath) Maybe it's just this typo that is causing the issue. On Fri, Jul 24, 2015 at 11:29 AM, wstoo1 [bioc] <noreply@bioconductor.org> wrote: > Activity on a post you are following on support.bioconductor.org > > User wstoo1 <https: support.bioconductor.org="" u="" 8468=""/> wrote Comment: > Failed to annotated my wells > <https: support.bioconductor.org="" p="" 70344="" #70346="">: > > Hi, > > The original script that my supervisor sent me was below: > > x <- readPlateList("platelist.txt", name = expName, path=dataPath) > x<-configure(x,confFile = confFile, logFile = logFile,descripFile = > desFile, path = dataPath) > #Calculate the survival ratio by normalizing FL channel to the RL > > xn<-summarizeChannels(x,fun = function (r1,r2,r3,r4,r5,r6,r7) r5/r3) > > #Normalise plates to the median of the plate values > > xnp <- normalizePlates(xn, scale = "multiplicative", log = TRUE, > method = "negatives", varianceAdjust = "none") > > #Score wells within each replicate by caluclating a z score for each well > based on replicate median and mad > xsc <- scoreReplicates(xnp, sign = "+", method = "zscore") > > #summarize the replicates by taking the mean value for each > xf <- summarizeReplicates(xsc, summary = "mean") > > xfw<-annotate(xf,geneIDFile="annotationST1.txt",path=dataPath) > > He annotated to xfw variable, which has scores. > > In the write report function, the scored input he used was xf. > > out <- writeReport(raw=x, normalized=xnp, scored=xf, force = TRUE, > plotPlateArgs = TRUE, imageScreenArgs = list(zrange=c( -4, 8),ar=1), > map=TRUE, outdir=outPath) > > Hence, the Annotated status was FALSE. > > However, no matter the annotated status in write report feedback is TRUE > or FALSE, the html report generated still no annotations to the wells. > > HELP !!!! > > ------------------------------ > > Post tags: cellhts2 > > You may reply via email or visit > C: Failed to annotated my wells >
ADD REPLYlink written 4.2 years ago by Joseph Barry160
Answer: Failed to annotated my wells
0
gravatar for wstoo1
4.2 years ago by
wstoo10
European Union
wstoo10 wrote:

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,

ADD COMMENTlink written 4.2 years ago by wstoo10
Answer: Failed to annotated my wells
0
gravatar for Joseph Barry
4.2 years ago by
Joseph Barry160
Dana-Farber Cancer Institute, Boston, USA
Joseph Barry160 wrote:

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.

ADD COMMENTlink written 4.2 years ago by Joseph Barry160

Yes. I tried to annotate my raw data x but nothing change in the html report.

The annotation status at the end was true. The html report didnt show the annotation correctly.

It showed:

WellAnno finalWellAnno

sample sample

It seems like the annotation function is working normally, but failed to link the write report function.

ADD REPLYlink written 4.2 years ago by wstoo10

Okay, at this point it is hard for me to pinpoint the problem without seeing the data. Would you mind privately sending me example data?  joseph.barry@... please get the rest from my user profile

ADD REPLYlink written 4.2 years ago by Joseph Barry160
Answer: Failed to annotated my wells
0
gravatar for Joseph Barry
4.1 years ago by
Joseph Barry160
Dana-Farber Cancer Institute, Boston, USA
Joseph Barry160 wrote:

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.

ADD COMMENTlink written 4.1 years ago by Joseph Barry160
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