how to check which probesets belong to which transcript?
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Biologist ▴ 120
@biologist-9801
Last seen 4.7 years ago

Im first time working on microarray data analysis. My chip is hugene 2.0 ST. Using xps package I have differential expressed exons.

   Probeset ID UNIT_ID Statistics    Mean1    Mean2 StandardError DegreeOfFreedom
    16657437    3742   -3.87699  1.80551  8.27607      0.566558               4
    16657441    3745    2.89796  3.81905  1.38638      0.504455               4
    16657456    3757    3.14543  4.51182  1.69900      0.447957               4
    16657519    3802   -3.04069  5.91567 18.16170      0.532210               4
    16657589    3857   -2.97385  4.26765 10.45620      0.434738               4
    16657604    3867   -3.26664 21.48650 37.10300      0.241258               4
   Probeset ID  P-Value P-Adjusted FoldChange
    16657437 0.0178869  0.0178869   4.583780
    16657441 0.0442085  0.0442085   0.363018
    16657456 0.0346659  0.0346659   0.376568
    16657519 0.0383762  0.0383762   3.070100
    16657589 0.0409876  0.0409876   2.450110
    16657604 0.0308867  0.0308867   1.726800

In the above table the probeset Id represents differential expressed exons. So now, How to check which probesets belong to which transcript? Thank you in advance.
xps affymetrix microarrays humangene2.0ST micro array data • 4.5k views
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cstrato ★ 3.9k
@cstrato-908
Last seen 6.1 years ago
Austria

The answer is shown in my reply to your question C: How to import normalized file (using xps) to limma for differential expressed ge
where column 'UnitName' is the probesetID and column 'TranscriptID' the transcriptID.

Regards,
Christian
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Thank you. Could you please tell me how to find the best probeset ?

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cstrato ★ 3.9k
@cstrato-908
Last seen 6.1 years ago
Austria

Maybe other experts do know a way, but I do not think that there is a general way to find the best
probeset for each transcript, especially since for many genes multiple transcripts do exist, due to 
alternative splicing, e.g. EGFR gene.  (For individual transcripts it is possible to look in the 
Ensemble Genome Browser, which probes are located within the exons of a gene.)

Regards,
Christian
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Hi christian,

data.rma <- rma(data.exon, "tmp_exonRMA", filedir=rootdir, verbose=FALSE, exonlevel="core", option="probeset", background="antigenomic")


expr.rma <- export.expr(data.rma, treename = "*", treetype = "mdp", varlist = "fUnitName:fTranscriptID:fLevel", outfile = "MyName.txt", sep = "\t", as.dataframe = FALSE, verbose = TRUE)

Is it possible to copy expr.rma into a dataframe or datatable?

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Please read the help '?export.expr' again!!    hint: look at parameter 'as.dataframe'

 

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Thank you. And Is it possible to create root for a dataframe?

unifltr <- UniFilter(unitest=c("t.test","two.sided","none",0,0.0,FALSE,0.95,TRUE), foldchange=c(1.5,"both"), unifilter=c(0.05,"pval"))

rma.ufr <- unifilter(data.rma, "tmp_exonUnifilter", filedir=rootdir , filter=unifltr, group=c("GrpA","GrpA","GrpA","GrpB","GrpB","GrpB"), verbose=FALSE)

I have my new normalized values in a dataframe. so how can I use unifilter for that ? @cstrato

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Maybe, it is possible, but you need to be careful, and you need to read the help '?exprs' carefully!

This is what you could do, but w/o guarantee:

# first check how 'data.rma' looks like
> str(data.rma)

# get expression values
> value <- exprs(data.rma)

# now modify 'value' so that it contains your data

# make a copy of your object if you do not want to replace it
> subset.rma <- data.rma

## replace slot data with the modified 'value'
> exprs(subset.rma) <- value
> str(subset.rma)

Regards,
Christian

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Ok I will try this. But data.rma doesnt have transcript ID. So can I use expr.rma (which have transcript ID) for unifilter? I want to do differential analysis with Transcript ID. @cstrato

can I do like this ?

> value <- exprs(expr.rma)

I tried in this way now

data.rma <- rma(data.exon, "tmp_exonRMA", filedir=rootdir, verbose=FALSE, exonlevel="core", option="probeset", background="antigenomic")

expr.rma <- export.expr(data.rma, treename = "*", treetype = "mdp", varlist = "fUnitName:fTranscriptID:fLevel", outfile = "MyName.txt", sep = "\t", as.dataframe = TRUE, verbose = TRUE)

Here expr.rma is a dataframe.

As in my samples Each transcript has multiple probesets so I want to calculate mean of the probesets.

library(data.table)
DT <- data.table(expr.rma)

value <- data.frame(DT[, sapply(.SD, function(x) list(mean=mean(x))), by=TranscriptID])

So now how to do unifilter for differential analysis?

unifltr <- UniFilter(unitest=c("t.test","two.sided","none",0,0.0,FALSE,0.95,TRUE), foldchange=c(1.5,"both"), unifilter=c(0.05,"pval"))

rma.ufr <- unifilter(data.rma, "tmp_exonUnifilter", filedir=rootdir , filter=unifltr, group=c("GrpA","GrpA","GrpA","GrpB","GrpB","GrpB"), logbase = log, verbose=FALSE)

 

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I don't know, but you can try it. Maybe it works, however, probably the rownames should be identical.

 

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BUt for unifilter what I need to give? 

rma.ufr <- unifilter(data.rma, "tmp_exonUnifilter", filedir=rootdir , filter=unifltr, group=c("GrpA","GrpA","GrpA","GrpB","GrpB","GrpB"), logbase = log, verbose=FALSE)

can I give value instead of data.rma? because I want to do difeerential analysis for "value" which has my changes

And is it possible to make a root for text file? @cstrato

When I gave this

> rma.ufr <- unifilter(value, "tmp_exonUnifilter", filedir=rootdir , filter=unifltr, group=c("GrpA","GrpA","GrpA","GrpB","GrpB","GrpB"), verbose=FALSE)

I got the following error:


Error in unifilter(value, "tmp_exonUnifilter", filedir = rootdir, filter = unifltr,  : 
  ‘xps.expr’ is not a class ‘ExprTreeSet’

so how to do this? please help me.

 

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You cannot use a data.frame, you have to do it as I have mentioned:

# get expression values
> value <- exprs(data.rma)

# now modify 'value' so that it contains your data

# make a copy of your object if you do not want to replace it
> subset.rma <- data.rma

## replace slot data with the modified 'value'
> exprs(subset.rma) <- value
> str(subset.rma)

If 'data.rma' does not have transcript ID but 'expr.rma' does have it, then you can 
add it later from 'expr.rma' to 'data.rma' or to the result you get.

Regards,
Christian

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Without Transcript ID I cannot make the changes. As I said before in my data each Transcript has multiple probesets so based on transcript id i want to calculate mean of the probesets and then give that to unifilter instead of data.rma Is there any other possible way to do differential analysis without unifilter? I cannot use limma because it does foldchange calculation in a different way and it gives log2 values. I need the log values.
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If you want to get the transcripts as mean from the probesets, then the simplest way is the following. 
Instead of:
> data.rma <- rma(data.exon, "tmp_exonRMA", filedir=rootdir, verbose=FALSE, exonlevel="core", option="probeset", background="antigenomic")
simply do:
> data.rma <- rma(data.exon, "tmp_exonRMA", filedir=rootdir, verbose=FALSE, exonlevel="core", option="transcript", background="antigenomic")
This you can pass immediately to unifilter.

Most people use 'limma'. 
I do not see any problem to convert log2 values to log values. This is basic R knowledge.
Please ask the 'R-help' mailing list if you do not know how to convert log2 to log.

Regards,
Christian

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In your comment value is a dataframe? Can I add a column Transcript ID to "value" from MyName.txt Is it possible to do that? @cstrato
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I added a column "TranscriptID" in datarame "value" from "MyName.txt". And calculated mean of probesets as there are mutiple probesets.

head(value)
  TranscriptID UNIT_ID.mean UnitName.mean
1     16657436       3743.0      16657438
2     16657440       3746.5      16657443
3     16657445       3749.0      16657446
4     16657447       3750.5      16657449
5     16657450       3760.5      16657460
6     16657469       3771.0      16657471
  X_8__HuGene_2_0_st_.mdp_LEVEL.mean
1                           5.323233
2                          14.718513
3                           1.684600
4                           2.178150
5                          28.057541
6                           2.499803
  E__HuGene_2_0_st_.mdp_LEVEL.mean
1                         3.767767
2                        10.625183
3                         1.403030
4                         1.572695
5                         8.954574
6                         2.429183
  GG7__HuGene_2_0_st_.mdp_LEVEL.mean
1                           4.989677
2                          10.204902
3                           1.445910
4                           1.533365
5                          29.077408
6                           5.008880
  J__HuGene_2_0_st_.mdp_LEVEL.mean O__HuGene_2_0_st_.mdp_LEVEL.mean
1                         5.474803                         9.415533
2                         5.588540                        13.181102
3                         2.181390                         2.370500
4                         2.297310                         2.355175
5                        17.902806                        12.137821
6                         3.587063                         2.787710
  T__HuGene_2_0_st_.mdp_LEVEL.mean
1                         7.715870
2                         6.288207
3                         5.623490
4                         1.486745
5                         8.019229
6                         3.240043

For differential analysis. I tried doing what you said.

# make a copy of your object if you do not want to replace it
> subset.rma <- data.rma

## replace slot data with the modified 'value'
> exprs(subset.rma) <- value
it gave me this error.

Error in .local(object, ..., value) : 
  argument "value" is missing, with no default

 

 

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To understand how 'value' should look like please run first the example given in the help for function 'exprs()'.

You are not allowed to rename columns "UNIT_ID" and "UnitName". You need to remove '.mean' from all column names.
Furthermore, "UNIT_ID" is an integer which you are not allowed to change! You cannot create a mean UNIT_ID!!

Regards,
Christian

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Hi Christian, 

As you said I tried it.

> str(data.rma)
Formal class 'ExprTreeSet' [package "xps"] with 11 slots
  ..@ exprtype : chr "rma"
  ..@ normtype : chr "none"
  ..@ scheme   :Formal class 'SchemeTreeSet' [package "xps"] with 12 slots
  .. .. ..@ chipname : chr "HuGene-2_0-st"
  .. .. ..@ chiptype : chr "ExonChip"
  .. .. ..@ probeinfo:List of 8
  .. .. .. ..$ nrows     : int 1612
  .. .. .. ..$ ncols     : int 1612
  .. .. .. ..$ nprobes   : int 1022045
  .. .. .. ..$ ncontrols : int 23
  .. .. .. ..$ ngenes    : int 48204
  .. .. .. ..$ nunits    : int 51991
  .. .. .. ..$ nprobesets: int 352859
  .. .. .. ..$ naffx     : int 167
  .. .. ..@ probe    :'data.frame':     0 obs. of  0 variables
  .. .. ..@ unitname :'data.frame':     0 obs. of  0 variables
  .. .. ..@ mask     :'data.frame':     0 obs. of  0 variables
  .. .. ..@ setname  : chr "HuGene-2_0-st"
  .. .. ..@ settype  : chr "scheme"
  .. .. ..@ rootfile : chr "D:\\GeneST_Analyse\\New4516/schemes/Scheme.root"
  .. .. ..@ filedir  : chr "D:\\GeneST_Analyse\\New4516/schemes"
  .. .. ..@ numtrees : int 9
  .. .. ..@ treenames:List of 9
  .. .. .. ..$ : chr "HuGene-2_0-st.cxy"
  .. .. .. ..$ : chr "HuGene-2_0-st.anx"
  .. .. .. ..$ : chr "HuGene-2_0-st.anp"
  .. .. .. ..$ : chr "HuGene-2_0-st.scm"
  .. .. .. ..$ : chr "HuGene-2_0-st.idx"
  .. .. .. ..$ : chr "HuGene-2_0-st.prb"
  .. .. .. ..$ : chr "HuGene-2_0-st.exn"
  .. .. .. ..$ : chr "HuGene-2_0-st.pbs"
  .. .. .. ..$ : chr "HuGene-2_0-st.ann"
  ..@ data     :'data.frame':   345497 obs. of  8 variables:
  .. ..$ UNIT_ID                      : int [1:345497] 3742 3743 3744 3745 3746 3747 3748 3749 3750 3751 ...
  .. ..$ UnitName                     : int [1:345497] 16657437 16657438 16657439 16657441 16657442 16657443 16657444 16657446 16657448 16657449 ...
  .. ..$ X_8__HuGene_2_0_st_.mdp_LEVEL: num [1:345497] 2.11 2.55 11.31 4.19 2.58 ...
  .. ..$ E__HuGene_2_0_st_.mdp_LEVEL  : num [1:345497] 2.11 1.92 7.27 6.3 1.89 ...
  .. ..$ GG7__HuGene_2_0_st_.mdp_LEVEL: num [1:345497] 1.32 3.63 10.01 2.11 1.88 ...
  .. ..$ J__HuGene_2_0_st_.mdp_LEVEL  : num [1:345497] 4.15 2.26 10.02 1.07 4.11 ...
  .. ..$ O__HuGene_2_0_st_.mdp_LEVEL  : num [1:345497] 9.77 3.25 15.23 1.43 3.59 ...
  .. ..$ T__HuGene_2_0_st_.mdp_LEVEL  : num [1:345497] 14 2.8 6.35 1.74 2.45 ...
  ..@ params   : list()
  ..@ setname  : chr "PreprocesSet"
  ..@ settype  : chr "preprocess"
  ..@ rootfile : chr "D:\\GeneST_Analyse\\New4516/rootdata/tmp_exonRMA.root"
  ..@ filedir  : chr "D:\\GeneST_Analyse\\New4516/rootdata"
  ..@ numtrees : int 6
  ..@ treenames:List of 6
  .. ..$ : chr "X_8__HuGene_2_0_st_.mdp"
  .. ..$ : chr "E__HuGene_2_0_st_.mdp"
  .. ..$ : chr "GG7__HuGene_2_0_st_.mdp"
  .. ..$ : chr "J__HuGene_2_0_st_.mdp"
  .. ..$ : chr "O__HuGene_2_0_st_.mdp"
  .. ..$ : chr "T__HuGene_2_0_st_.mdp"

value3 <- exprs(data.rma)

head(value3)
  UNIT_ID UnitName X_8__HuGene_2_0_st_.mdp_LEVEL
1    3742 16657437                       2.10763
2    3743 16657438                       2.54837
3    3744 16657439                      11.31370
4    3745 16657441                       4.19212
5    3746 16657442                       2.57503
6    3747 16657443                      41.90180
  E__HuGene_2_0_st_.mdp_LEVEL GG7__HuGene_2_0_st_.mdp_LEVEL
1                     2.10763                       1.32498
2                     1.92386                       3.63425
3                     7.27181                      10.00980
4                     6.30431                       2.10763
5                     1.89032                       1.87688
6                    22.00250                      23.64940
  J__HuGene_2_0_st_.mdp_LEVEL O__HuGene_2_0_st_.mdp_LEVEL
1                     4.14518                     9.77053
2                     2.26163                     3.24937
3                    10.01760                    15.22670
4                     1.06896                     1.43262
5                     4.10860                     3.58509
6                    10.64590                    29.45960
  T__HuGene_2_0_st_.mdp_LEVEL
1                    13.99620
2                     2.79736
3                     6.35405
4                     1.74003
5                     2.44511
6                    13.76480

subset.rma <- data.rma

exprs(subset.rma) <- value3
Error in .local(object, ..., value) : 
  argument "value" is missing, with no default

The same error. Could you please help me? @cstrato

 

 

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Since you have done:
> value3 <- exprs(data.rma)
you need to do:
> exprs(subset.rma) <- value3

Are you sure that you have used 'value3', since in the mail which informed me of a 
new 'activity on a post' I received the following:

....

subset.rma <- data.rma

exprs(subset.rma) <- value
Error in .local(object, ..., value) : 
  argument "value" is missing, with no default

The same error. Could you please help me?

Regards,
Christian

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No that is by mistake, its is value3

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See this Christian.

> data.rma <- rma(data.exon, "tmp_exonRMA", filedir=rootdir, verbose=FALSE, exonlevel="core", option="probeset", background="antigenomic")
> str(data.rma)
Formal class 'ExprTreeSet' [package "xps"] with 11 slots
  ..@ exprtype : chr "rma"
  ..@ normtype : chr "none"
  ..@ scheme   :Formal class 'SchemeTreeSet' [package "xps"] with 12 slots
  .. .. ..@ chipname : chr "HuGene-2_0-st"
  .. .. ..@ chiptype : chr "ExonChip"
  .. .. ..@ probeinfo:List of 8
  .. .. .. ..$ nrows     : int 1612
  .. .. .. ..$ ncols     : int 1612
  .. .. .. ..$ nprobes   : int 1022045
  .. .. .. ..$ ncontrols : int 23
  .. .. .. ..$ ngenes    : int 48204
  .. .. .. ..$ nunits    : int 51991
  .. .. .. ..$ nprobesets: int 352859
  .. .. .. ..$ naffx     : int 167
  .. .. ..@ probe    :'data.frame':     0 obs. of  0 variables
  .. .. ..@ unitname :'data.frame':     0 obs. of  0 variables
  .. .. ..@ mask     :'data.frame':     0 obs. of  0 variables
  .. .. ..@ setname  : chr "HuGene-2_0-st"
  .. .. ..@ settype  : chr "scheme"
  .. .. ..@ rootfile : chr "D:\\GeneST_Analyse\\New4516/schemes/Scheme.root"
  .. .. ..@ filedir  : chr "D:\\GeneST_Analyse\\New4516/schemes"
  .. .. ..@ numtrees : int 9
  .. .. ..@ treenames:List of 9
  .. .. .. ..$ : chr "HuGene-2_0-st.cxy"
  .. .. .. ..$ : chr "HuGene-2_0-st.anx"
  .. .. .. ..$ : chr "HuGene-2_0-st.anp"
  .. .. .. ..$ : chr "HuGene-2_0-st.scm"
  .. .. .. ..$ : chr "HuGene-2_0-st.idx"
  .. .. .. ..$ : chr "HuGene-2_0-st.prb"
  .. .. .. ..$ : chr "HuGene-2_0-st.exn"
  .. .. .. ..$ : chr "HuGene-2_0-st.pbs"
  .. .. .. ..$ : chr "HuGene-2_0-st.ann"
  ..@ data     :'data.frame':   345497 obs. of  8 variables:
  .. ..$ UNIT_ID                      : int [1:345497] 3742 3743 3744 3745 3746 3747 3748 3749 3750 3751 ...
  .. ..$ UnitName                     : int [1:345497] 16657437 16657438 16657439 16657441 16657442 16657443 16657444 16657446 16657448 16657449 ...
  .. ..$ X_8__HuGene_2_0_st_.mdp_LEVEL: num [1:345497] 2.11 2.55 11.31 4.19 2.58 ...
  .. ..$ E__HuGene_2_0_st_.mdp_LEVEL  : num [1:345497] 2.11 1.92 7.27 6.3 1.89 ...
  .. ..$ GG7__HuGene_2_0_st_.mdp_LEVEL: num [1:345497] 1.32 3.63 10.01 2.11 1.88 ...
  .. ..$ J__HuGene_2_0_st_.mdp_LEVEL  : num [1:345497] 4.15 2.26 10.02 1.07 4.11 ...
  .. ..$ O__HuGene_2_0_st_.mdp_LEVEL  : num [1:345497] 9.77 3.25 15.23 1.43 3.59 ...
  .. ..$ T__HuGene_2_0_st_.mdp_LEVEL  : num [1:345497] 14 2.8 6.35 1.74 2.45 ...
  ..@ params   : list()
  ..@ setname  : chr "PreprocesSet"
  ..@ settype  : chr "preprocess"
  ..@ rootfile : chr "D:\\GeneST_Analyse\\New4516/rootdata/tmp_exonRMA.root"
  ..@ filedir  : chr "D:\\GeneST_Analyse\\New4516/rootdata"
  ..@ numtrees : int 6
  ..@ treenames:List of 6
  .. ..$ : chr "X_8__HuGene_2_0_st_.mdp"
  .. ..$ : chr "E__HuGene_2_0_st_.mdp"
  .. ..$ : chr "GG7__HuGene_2_0_st_.mdp"
  .. ..$ : chr "J__HuGene_2_0_st_.mdp"
  .. ..$ : chr "O__HuGene_2_0_st_.mdp"
  .. ..$ : chr "T__HuGene_2_0_st_.mdp"
> val <- exprs(data.rma)
> head(val)
  UNIT_ID UnitName X_8__HuGene_2_0_st_.mdp_LEVEL
1    3742 16657437                       2.10763
2    3743 16657438                       2.54837
3    3744 16657439                      11.31370
4    3745 16657441                       4.19212
5    3746 16657442                       2.57503
6    3747 16657443                      41.90180
  E__HuGene_2_0_st_.mdp_LEVEL GG7__HuGene_2_0_st_.mdp_LEVEL
1                     2.10763                       1.32498
2                     1.92386                       3.63425
3                     7.27181                      10.00980
4                     6.30431                       2.10763
5                     1.89032                       1.87688
6                    22.00250                      23.64940
  J__HuGene_2_0_st_.mdp_LEVEL O__HuGene_2_0_st_.mdp_LEVEL
1                     4.14518                     9.77053
2                     2.26163                     3.24937
3                    10.01760                    15.22670
4                     1.06896                     1.43262
5                     4.10860                     3.58509
6                    10.64590                    29.45960
  T__HuGene_2_0_st_.mdp_LEVEL
1                    13.99620
2                     2.79736
3                     6.35405
4                     1.74003
5                     2.44511
6                    13.76480
> subset.rma <- data.rma
> exprs(subset.rma) <- val
Error in .local(object, ..., value) : 
  argument "value" is missing, with no default

 

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0
Entering edit mode

I am sorry, you need to do:

> val <- exprs(data.rma)
> subset.rma <- data.rma
> exprs(subset.rma, NULL) <- val

(When I had tested it yesterday, I did not have to add parameter NULL)

Regards,
Christian

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0
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Ok. Thank you. But Can I try subset.rma for unifilter? Like this

unifltr <- UniFilter(unitest=c("t.test","two.sided","none",0,0.0,FALSE,0.95,TRUE), foldchange=c(1.5,"both"), unifilter=c(0.05,"pval"))

rma.ufr <- unifilter(subset.rma, "tmp_exonUnifilter", filedir=rootdir , filter=unifltr, group=c("GrpA","GrpA","GrpA","GrpB","GrpB","GrpB"), verbose=FALSE)

ADD REPLY •
0
Entering edit mode

Since 'subset.rma' is of class ExprTreeSet it should be possible, however if you intend to modify the dataframe 'value' it depends what you do.

BTW, I have a question, too:
Why don't you simply try it yourself instead of asking whether it will work?

It takes you about one minute to see whether it works, but it will take from a few 
hours till one or more days to wait for an answer from me. (Currently I will not be 
available for the next few hours.)

Regards,
Christian

 

ADD REPLY •
0
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Yes, what you said was right. But I'm in home and I don't have a R 32 bit and obviously I cannot install root for xps. As you are giving reply only in the evenings I asked now itself. Please don't mind Thank you.

ADD REPLY •
0
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I understand. However, it should not be a problem to install ROOT. First, there is a README file where I explain how to install ROOT. Second, you can also ask these questions and I will try to guide you.

First I would need to know which computer you have at home, and what your 'sessionInfo()' is. And as far as I know, R works both with 32 bit and with 64 bit.

Regards,
Christian

 

ADD REPLY •
0
Entering edit mode

Thank you. I have a small question. unifilter produces log2FC values but I need normal fold change. How can I do that with unifilter function. Looking forward to your response.

ADD REPLY •
0
Entering edit mode

unifilter produces log2FC, you need to convert it yourself. See basic R help '?log2':   2^(log2(34.6)) !!!

 

ADD REPLY •
0
Entering edit mode

Sorry, my mistake: look at parameter 'logbase' of function 'unifilter()'.

ADD REPLY •
0
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Yes I saw. There is logbase 0, log2, log10. Is logbase = 0 normal fold change? It is not working.

ADD REPLY •
0
Entering edit mode

You must write: logbase ="0"

ADD REPLY •
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Entering edit mode

Hi Christian,

Again I need your help.

head(New)
  UNIT_ID UnitName X_8.mdp_LEVEL E.mdp_LEVEL GG7.mdp_LEVEL J.mdp_LEVEL
1    3742 16657437       2.10763     2.10763       1.32498     4.14518
2    3743 16657438       2.54837     1.92386       3.63425     2.26163
3    3744 16657439      11.31370     7.27181      10.00980    10.01760
4    3745 16657441       4.19212     6.30431       2.10763     1.06896
6    3747 16657443      41.90180    22.00250      23.64940    10.64590
7    3748 16657444      10.20510    12.30360      13.18570     6.53070
  O.mdp_LEVEL T.mdp_LEVEL
1     9.77053    13.99620
2     3.24937     2.79736
3    15.22670     6.35405
4     1.43262     1.74003
6    29.45960    13.76480
7    18.24710     7.20289

subset3.rma <- data.rma

exprs(subset3.rma, NULL) <- New

str(subset3.rma)

Formal class 'ExprTreeSet' [package "xps"] with 11 slots
  ..@ exprtype : chr "rma"
  ..@ normtype : chr "none"
  ..@ scheme   :Formal class 'SchemeTreeSet' [package "xps"] with 12 slots
  .. .. ..@ chipname : chr "HuGene-2_0-st"
  .. .. ..@ chiptype : chr "ExonChip"
  .. .. ..@ probeinfo:List of 8
  .. .. .. ..$ nrows     : int 1612
  .. .. .. ..$ ncols     : int 1612
  .. .. .. ..$ nprobes   : int 1022045
  .. .. .. ..$ ncontrols : int 23
  .. .. .. ..$ ngenes    : int 48204
  .. .. .. ..$ nunits    : int 51991
  .. .. .. ..$ nprobesets: int 352859
  .. .. .. ..$ naffx     : int 167
  .. .. ..@ probe    :'data.frame':     0 obs. of  0 variables
  .. .. ..@ unitname :'data.frame':     0 obs. of  0 variables
  .. .. ..@ mask     :'data.frame':     0 obs. of  0 variables
  .. .. ..@ setname  : chr "HuGene-2_0-st"
  .. .. ..@ settype  : chr "scheme"
  .. .. ..@ rootfile : chr "D:\\GeneST_Analyse\\New4516/schemes/Scheme.root"
  .. .. ..@ filedir  : chr "D:\\GeneST_Analyse\\New4516/schemes"
  .. .. ..@ numtrees : int 9
  .. .. ..@ treenames:List of 9
  .. .. .. ..$ : chr "HuGene-2_0-st.cxy"
  .. .. .. ..$ : chr "HuGene-2_0-st.anx"
  .. .. .. ..$ : chr "HuGene-2_0-st.anp"
  .. .. .. ..$ : chr "HuGene-2_0-st.scm"
  .. .. .. ..$ : chr "HuGene-2_0-st.idx"
  .. .. .. ..$ : chr "HuGene-2_0-st.prb"
  .. .. .. ..$ : chr "HuGene-2_0-st.exn"
  .. .. .. ..$ : chr "HuGene-2_0-st.pbs"
  .. .. .. ..$ : chr "HuGene-2_0-st.ann"
  ..@ data     :'data.frame':   106090 obs. of  8 variables:
  .. ..$ UNIT_ID      : int [1:106090] 3742 3743 3744 3745 3747 3748 3749 3750 3751 3752 ...
  .. ..$ UnitName     : int [1:106090] 16657437 16657438 16657439 16657441 16657443 16657444 16657446 16657448 16657449 16657451 ...
  .. ..$ X_8.mdp_LEVEL: num [1:106090] 2.11 2.55 11.31 4.19 41.9 ...
  .. ..$ E.mdp_LEVEL  : num [1:106090] 2.11 1.92 7.27 6.3 22 ...
  .. ..$ GG7.mdp_LEVEL: num [1:106090] 1.32 3.63 10.01 2.11 23.65 ...
  .. ..$ J.mdp_LEVEL  : num [1:106090] 4.15 2.26 10.02 1.07 10.65 ...
  .. ..$ O.mdp_LEVEL  : num [1:106090] 9.77 3.25 15.23 1.43 29.46 ...
  .. ..$ T.mdp_LEVEL  : num [1:106090] 14 2.8 6.35 1.74 13.76 ...
  ..@ params   : list()
  ..@ setname  : chr "PreprocesSet"
  ..@ settype  : chr "preprocess"
  ..@ rootfile : chr "D:\\GeneST_Analyse\\New4516/rootdata/tmp_exonRMA.root"
  ..@ filedir  : chr "D:\\GeneST_Analyse\\New4516/rootdata"
  ..@ numtrees : int 6
  ..@ treenames:List of 6
  .. ..$ : chr "X_8.mdp"
  .. ..$ : chr "E.mdp"
  .. ..$ : chr "GG7.mdp"
  .. ..$ : chr "J.mdp"
  .. ..$ : chr "O.mdp"
  .. ..$ : chr "T.mdp"

Here in this there are totally 10,691 probesets.

Till here it is fine. But after this I want to do Differential analysis. So using unifilter function.

unifltr <- UniFilter(unitest=c("t.test","two.sided","none",0,0.0,FALSE,0.95,TRUE), foldchange=c(1.5,"both"), unifilter=c(0.05,"pval"))

rma.ufr <- unifilter(subset3.rma, "tmp_exonUnifilter", filedir=rootdir , filter=unifltr, logbase = "0", group=c("GrpA","GrpA","GrpA","GrpB","GrpB","GrpB"), verbose=FALSE)
tmp <- validData(rma.ufr)
dim(tmp)

[1] 11085     9

Formal class 'AnalysisTreeSet' [package "xps"] with 10 slots
  ..@ fltrset  :Formal class 'FilterTreeSet' [package "xps"] with 12 slots
  .. .. ..@ filter   :Formal class 'UniFilter' [package "xps"] with 5 slots
  .. .. .. .. ..@ foldchange:List of 2
  .. .. .. .. .. ..$ cutoff   : num 1.5
  .. .. .. .. .. ..$ direction: chr "both"
  .. .. .. .. ..@ prescall  : list()
  .. .. .. .. ..@ unifilter :List of 2
  .. .. .. .. .. ..$ cutoff  : num 0.05
  .. .. .. .. .. ..$ variable: chr "pval"
  .. .. .. .. ..@ unitest   :List of 8
  .. .. .. .. .. ..$ type       : chr "t.test"
  .. .. .. .. .. ..$ alternative: chr "two.sided"
  .. .. .. .. .. ..$ correction : chr "none"
  .. .. .. .. .. ..$ numperm    : int 0
  .. .. .. .. .. ..$ mu         : num 0
  .. .. .. .. .. ..$ paired     : logi FALSE
  .. .. .. .. .. ..$ conflevel  : num 0.95
  .. .. .. .. .. ..$ varequ     : logi TRUE
  .. .. .. .. ..@ numfilters: num 2
  .. .. ..@ exprset  :Formal class 'ExprTreeSet' [package "xps"] with 11 slots
  .. .. .. .. ..@ exprtype : chr "rma"
  .. .. .. .. ..@ normtype : chr "none"
  .. .. .. .. ..@ scheme   :Formal class 'SchemeTreeSet' [package "xps"] with 12 slots
  .. .. .. .. .. .. ..@ chipname : chr "HuGene-2_0-st"
  .. .. .. .. .. .. ..@ chiptype : chr "ExonChip"
  .. .. .. .. .. .. ..@ probeinfo:List of 8
  .. .. .. .. .. .. .. ..$ nrows     : int 1612
  .. .. .. .. .. .. .. ..$ ncols     : int 1612
  .. .. .. .. .. .. .. ..$ nprobes   : int 1022045
  .. .. .. .. .. .. .. ..$ ncontrols : int 23
  .. .. .. .. .. .. .. ..$ ngenes    : int 48204
  .. .. .. .. .. .. .. ..$ nunits    : int 51991
  .. .. .. .. .. .. .. ..$ nprobesets: int 352859
  .. .. .. .. .. .. .. ..$ naffx     : int 167
  .. .. .. .. .. .. ..@ probe    :'data.frame': 0 obs. of  0 variables
  .. .. .. .. .. .. ..@ unitname :'data.frame': 0 obs. of  0 variables
  .. .. .. .. .. .. ..@ mask     :'data.frame': 0 obs. of  0 variables
  .. .. .. .. .. .. ..@ setname  : chr "HuGene-2_0-st"
  .. .. .. .. .. .. ..@ settype  : chr "scheme"
  .. .. .. .. .. .. ..@ rootfile : chr "D:\\GeneST_Analyse\\New4516/schemes/Scheme.root"
  .. .. .. .. .. .. ..@ filedir  : chr "D:\\GeneST_Analyse\\New4516/schemes"
  .. .. .. .. .. .. ..@ numtrees : int 9
  .. .. .. .. .. .. ..@ treenames:List of 9
  .. .. .. .. .. .. .. ..$ : chr "HuGene-2_0-st.cxy"
  .. .. .. .. .. .. .. ..$ : chr "HuGene-2_0-st.anx"
  .. .. .. .. .. .. .. ..$ : chr "HuGene-2_0-st.anp"
  .. .. .. .. .. .. .. ..$ : chr "HuGene-2_0-st.scm"
  .. .. .. .. .. .. .. ..$ : chr "HuGene-2_0-st.idx"
  .. .. .. .. .. .. .. ..$ : chr "HuGene-2_0-st.prb"
  .. .. .. .. .. .. .. ..$ : chr "HuGene-2_0-st.exn"
  .. .. .. .. .. .. .. ..$ : chr "HuGene-2_0-st.pbs"
  .. .. .. .. .. .. .. ..$ : chr "HuGene-2_0-st.ann"
  .. .. .. .. ..@ data     :'data.frame':       106090 obs. of  8 variables:
  .. .. .. .. .. ..$ UNIT_ID      : int [1:106090] 3742 3743 3744 3745 3747 3748 3749 3750 3751 3752 ...
  .. .. .. .. .. ..$ UnitName     : int [1:106090] 16657437 16657438 16657439 16657441 16657443 16657444 16657446 16657448 16657449 16657451 ...
  .. .. .. .. .. ..$ X_8.mdp_LEVEL: num [1:106090] 2.11 2.55 11.31 4.19 41.9 ...
  .. .. .. .. .. ..$ E.mdp_LEVEL  : num [1:106090] 2.11 1.92 7.27 6.3 22 ...
  .. .. .. .. .. ..$ GG7.mdp_LEVEL: num [1:106090] 1.32 3.63 10.01 2.11 23.65 ...
  .. .. .. .. .. ..$ J.mdp_LEVEL  : num [1:106090] 4.15 2.26 10.02 1.07 10.65 ...
  .. .. .. .. .. ..$ O.mdp_LEVEL  : num [1:106090] 9.77 3.25 15.23 1.43 29.46 ...
  .. .. .. .. .. ..$ T.mdp_LEVEL  : num [1:106090] 14 2.8 6.35 1.74 13.76 ...
  .. .. .. .. ..@ params   : list()
  .. .. .. .. ..@ setname  : chr "PreprocesSet"
  .. .. .. .. ..@ settype  : chr "preprocess"
  .. .. .. .. ..@ rootfile : chr "D:\\GeneST_Analyse\\New4516/rootdata/tmp_exonRMA.root"
  .. .. .. .. ..@ filedir  : chr "D:\\GeneST_Analyse\\New4516/rootdata"
  .. .. .. .. ..@ numtrees : int 6
  .. .. .. .. ..@ treenames:List of 6
  .. .. .. .. .. ..$ : chr "X_8.mdp"
  .. .. .. .. .. ..$ : chr "E.mdp"
  .. .. .. .. .. ..$ : chr "GG7.mdp"
  .. .. .. .. .. ..$ : chr "J.mdp"
  .. .. .. .. .. ..$ : chr "O.mdp"
  .. .. .. .. .. ..$ : chr "T.mdp"
  .. .. ..@ callset  :Formal class 'CallTreeSet' [package "xps"] with 11 slots
  .. .. .. .. ..@ calltype : chr "mas5"
  .. .. .. .. ..@ detcall  :'data.frame':       0 obs. of  0 variables
  .. .. .. .. ..@ scheme   :Formal class 'SchemeTreeSet' [package "xps"] with 12 slots
  .. .. .. .. .. .. ..@ chipname : chr ""
  .. .. .. .. .. .. ..@ chiptype : chr "GeneChip"
  .. .. .. .. .. .. ..@ probeinfo: list()
  .. .. .. .. .. .. ..@ probe    :'data.frame': 0 obs. of  0 variables
  .. .. .. .. .. .. ..@ unitname :'data.frame': 0 obs. of  0 variables
  .. .. .. .. .. .. ..@ mask     :'data.frame': 0 obs. of  0 variables
  .. .. .. .. .. .. ..@ setname  : chr ""
  .. .. .. .. .. .. ..@ settype  : chr ""
  .. .. .. .. .. .. ..@ rootfile : chr "ROOTFile"
  .. .. .. .. .. .. ..@ filedir  : chr "C:/biocbld/bbs-3.2-bioc/tmpdir/Rtmpy2DVDR/R.INSTALLc98a4d4a9/xps"
  .. .. .. .. .. .. ..@ numtrees : num 0
  .. .. .. .. .. .. ..@ treenames: list()
  .. .. .. .. ..@ data     :'data.frame':       0 obs. of  0 variables
  .. .. .. .. ..@ params   : list()
  .. .. .. .. ..@ setname  : chr ""
  .. .. .. .. ..@ settype  : chr ""
  .. .. .. .. ..@ rootfile : chr "ROOTFile"
  .. .. .. .. ..@ filedir  : chr "C:/biocbld/bbs-3.2-bioc/tmpdir/Rtmpy2DVDR/R.INSTALLc98a4d4a9/xps"
  .. .. .. .. ..@ numtrees : num 0
  .. .. .. .. ..@ treenames: list()
  .. .. ..@ scheme   :Formal class 'SchemeTreeSet' [package "xps"] with 12 slots
  .. .. .. .. ..@ chipname : chr "HuGene-2_0-st"
  .. .. .. .. ..@ chiptype : chr "ExonChip"
  .. .. .. .. ..@ probeinfo:List of 8
  .. .. .. .. .. ..$ nrows     : int 1612
  .. .. .. .. .. ..$ ncols     : int 1612
  .. .. .. .. .. ..$ nprobes   : int 1022045
  .. .. .. .. .. ..$ ncontrols : int 23
  .. .. .. .. .. ..$ ngenes    : int 48204
  .. .. .. .. .. ..$ nunits    : int 51991
  .. .. .. .. .. ..$ nprobesets: int 352859
  .. .. .. .. .. ..$ naffx     : int 167
  .. .. .. .. ..@ probe    :'data.frame':       0 obs. of  0 variables
  .. .. .. .. ..@ unitname :'data.frame':       0 obs. of  0 variables
  .. .. .. .. ..@ mask     :'data.frame':       0 obs. of  0 variables
  .. .. .. .. ..@ setname  : chr "HuGene-2_0-st"
  .. .. .. .. ..@ settype  : chr "scheme"
  .. .. .. .. ..@ rootfile : chr "D:\\GeneST_Analyse\\New4516/schemes/Scheme.root"
  .. .. .. .. ..@ filedir  : chr "D:\\GeneST_Analyse\\New4516/schemes"
  .. .. .. .. ..@ numtrees : int 9
  .. .. .. .. ..@ treenames:List of 9
  .. .. .. .. .. ..$ : chr "HuGene-2_0-st.cxy"
  .. .. .. .. .. ..$ : chr "HuGene-2_0-st.anx"
  .. .. .. .. .. ..$ : chr "HuGene-2_0-st.anp"
  .. .. .. .. .. ..$ : chr "HuGene-2_0-st.scm"
  .. .. .. .. .. ..$ : chr "HuGene-2_0-st.idx"
  .. .. .. .. .. ..$ : chr "HuGene-2_0-st.prb"
  .. .. .. .. .. ..$ : chr "HuGene-2_0-st.exn"
  .. .. .. .. .. ..$ : chr "HuGene-2_0-st.pbs"
  .. .. .. .. .. ..$ : chr "HuGene-2_0-st.ann"
  .. .. ..@ data     :'data.frame':     345497 obs. of  3 variables:
  .. .. .. ..$ UNIT_ID : int [1:345497] 3742 3743 3744 3745 3746 3747 3748 3749 3750 3751 ...
  .. .. .. ..$ UnitName: int [1:345497] 16657437 16657438 16657439 16657441 16657442 16657443 16657444 16657446 16657448 16657449 ...
  .. .. .. ..$ FLAG    : int [1:345497] 0 0 0 0 0 0 0 0 0 0 ...
  .. .. ..@ params   : list()
  .. .. ..@ setname  : chr "UniFilterSet"
  .. .. ..@ settype  : chr "unifilter"
  .. .. ..@ rootfile : chr "D:\\GeneST_Analyse\\New4516/rootdata/tmp_exonUnifilter_ufr.root"
  .. .. ..@ filedir  : chr "D:\\GeneST_Analyse\\New4516/rootdata"
  .. .. ..@ numtrees : int 1
  .. .. ..@ treenames:List of 1
  .. .. .. ..$ : chr "UniTest.ufr"
  ..@ scheme   :Formal class 'SchemeTreeSet' [package "xps"] with 12 slots
  .. .. ..@ chipname : chr "HuGene-2_0-st"
  .. .. ..@ chiptype : chr "ExonChip"
  .. .. ..@ probeinfo:List of 8
  .. .. .. ..$ nrows     : int 1612
  .. .. .. ..$ ncols     : int 1612
  .. .. .. ..$ nprobes   : int 1022045
  .. .. .. ..$ ncontrols : int 23
  .. .. .. ..$ ngenes    : int 48204
  .. .. .. ..$ nunits    : int 51991
  .. .. .. ..$ nprobesets: int 352859
  .. .. .. ..$ naffx     : int 167
  .. .. ..@ probe    :'data.frame':     0 obs. of  0 variables
  .. .. ..@ unitname :'data.frame':     0 obs. of  0 variables
  .. .. ..@ mask     :'data.frame':     0 obs. of  0 variables
  .. .. ..@ setname  : chr "HuGene-2_0-st"
  .. .. ..@ settype  : chr "scheme"
  .. .. ..@ rootfile : chr "D:\\GeneST_Analyse\\New4516/schemes/Scheme.root"
  .. .. ..@ filedir  : chr "D:\\GeneST_Analyse\\New4516/schemes"
  .. .. ..@ numtrees : int 9
  .. .. ..@ treenames:List of 9
  .. .. .. ..$ : chr "HuGene-2_0-st.cxy"
  .. .. .. ..$ : chr "HuGene-2_0-st.anx"
  .. .. .. ..$ : chr "HuGene-2_0-st.anp"
  .. .. .. ..$ : chr "HuGene-2_0-st.scm"
  .. .. .. ..$ : chr "HuGene-2_0-st.idx"
  .. .. .. ..$ : chr "HuGene-2_0-st.prb"
  .. .. .. ..$ : chr "HuGene-2_0-st.exn"
  .. .. .. ..$ : chr "HuGene-2_0-st.pbs"
  .. .. .. ..$ : chr "HuGene-2_0-st.ann"
  ..@ data     :'data.frame':   345497 obs. of  10 variables:
  .. ..$ UNIT_ID        : int [1:345497] 3742 3743 3744 3745 3746 3747 3748 3749 3750 3751 ...
  .. ..$ UnitName       : int [1:345497] 16657437 16657438 16657439 16657441 16657442 16657443 16657444 16657446 16657448 16657449 ...
  .. ..$ Statistics     : num [1:345497] -2.603 -0.117 -0.353 2.272 -2.333 ...
  .. ..$ Mean1          : num [1:345497] 1.85 2.7 9.53 4.2 2.11 ...
  .. ..$ Mean2          : num [1:345497] 9.3 2.77 10.53 1.41 3.38 ...
  .. ..$ StandardError  : num [1:345497] 2.865 0.575 2.836 1.227 0.542 ...
  .. ..$ DegreeOfFreedom: int [1:345497] 4 4 4 4 4 4 4 4 4 4 ...
  .. ..$ P-Value        : num [1:345497] 0.0599 0.9125 0.742 0.0855 0.08 ...
  .. ..$ P-Adjusted     : num [1:345497] 0.0599 0.9125 0.742 0.0855 0.08 ...
  .. ..$ FoldChange     : num [1:345497] 5.038 1.025 1.105 0.337 1.599 ...
  ..@ params   : list()
  ..@ setname  : chr "UniFilterSet"
  ..@ settype  : chr "UnivariateAnalysis"
  ..@ rootfile : chr "D:\\GeneST_Analyse\\New4516/rootdata/tmp_exonUnifilter_ufr.root"
  ..@ filedir  : chr "D:\\GeneST_Analyse\\New4516/rootdata"
  ..@ numtrees : int 1
  ..@ treenames:List of 1
  .. ..$ : chr "UniTest.stt"
> unifltr <- UniFilter(unitest=c("t.test","two.sided","none",0,0.0,FALSE,0.95,TRUE), foldchange=c(1.5,"both"), unifilter=c(0.05,"pval"))
> rma.ufr <- unifilter(subset3.rma, "tmp_exonUnifilter", filedir=rootdir , filter=unifltr, logbase = "0", group=c("GrpA","GrpA","GrpA","GrpB","GrpB","GrpB"), verbose=FALSE)
> str(rma.ufr)
Formal class 'AnalysisTreeSet' [package "xps"] with 10 slots
  ..@ fltrset  :Formal class 'FilterTreeSet' [package "xps"] with 12 slots
  .. .. ..@ filter   :Formal class 'UniFilter' [package "xps"] with 5 slots
  .. .. .. .. ..@ foldchange:List of 2
  .. .. .. .. .. ..$ cutoff   : num 1.5
  .. .. .. .. .. ..$ direction: chr "both"
  .. .. .. .. ..@ prescall  : list()
  .. .. .. .. ..@ unifilter :List of 2
  .. .. .. .. .. ..$ cutoff  : num 0.05
  .. .. .. .. .. ..$ variable: chr "pval"
  .. .. .. .. ..@ unitest   :List of 8
  .. .. .. .. .. ..$ type       : chr "t.test"
  .. .. .. .. .. ..$ alternative: chr "two.sided"
  .. .. .. .. .. ..$ correction : chr "none"
  .. .. .. .. .. ..$ numperm    : int 0
  .. .. .. .. .. ..$ mu         : num 0
  .. .. .. .. .. ..$ paired     : logi FALSE
  .. .. .. .. .. ..$ conflevel  : num 0.95
  .. .. .. .. .. ..$ varequ     : logi TRUE
  .. .. .. .. ..@ numfilters: num 2
  .. .. ..@ exprset  :Formal class 'ExprTreeSet' [package "xps"] with 11 slots
  .. .. .. .. ..@ exprtype : chr "rma"
  .. .. .. .. ..@ normtype : chr "none"
  .. .. .. .. ..@ scheme   :Formal class 'SchemeTreeSet' [package "xps"] with 12 slots
  .. .. .. .. .. .. ..@ chipname : chr "HuGene-2_0-st"
  .. .. .. .. .. .. ..@ chiptype : chr "ExonChip"
  .. .. .. .. .. .. ..@ probeinfo:List of 8
  .. .. .. .. .. .. .. ..$ nrows     : int 1612
  .. .. .. .. .. .. .. ..$ ncols     : int 1612
  .. .. .. .. .. .. .. ..$ nprobes   : int 1022045
  .. .. .. .. .. .. .. ..$ ncontrols : int 23
  .. .. .. .. .. .. .. ..$ ngenes    : int 48204
  .. .. .. .. .. .. .. ..$ nunits    : int 51991
  .. .. .. .. .. .. .. ..$ nprobesets: int 352859
  .. .. .. .. .. .. .. ..$ naffx     : int 167
  .. .. .. .. .. .. ..@ probe    :'data.frame': 0 obs. of  0 variables
  .. .. .. .. .. .. ..@ unitname :'data.frame': 0 obs. of  0 variables
  .. .. .. .. .. .. ..@ mask     :'data.frame': 0 obs. of  0 variables
  .. .. .. .. .. .. ..@ setname  : chr "HuGene-2_0-st"
  .. .. .. .. .. .. ..@ settype  : chr "scheme"
  .. .. .. .. .. .. ..@ rootfile : chr "D:\\GeneST_Analyse\\New4516/schemes/Scheme.root"
  .. .. .. .. .. .. ..@ filedir  : chr "D:\\GeneST_Analyse\\New4516/schemes"
  .. .. .. .. .. .. ..@ numtrees : int 9
  .. .. .. .. .. .. ..@ treenames:List of 9
  .. .. .. .. .. .. .. ..$ : chr "HuGene-2_0-st.cxy"
  .. .. .. .. .. .. .. ..$ : chr "HuGene-2_0-st.anx"
  .. .. .. .. .. .. .. ..$ : chr "HuGene-2_0-st.anp"
  .. .. .. .. .. .. .. ..$ : chr "HuGene-2_0-st.scm"
  .. .. .. .. .. .. .. ..$ : chr "HuGene-2_0-st.idx"
  .. .. .. .. .. .. .. ..$ : chr "HuGene-2_0-st.prb"
  .. .. .. .. .. .. .. ..$ : chr "HuGene-2_0-st.exn"
  .. .. .. .. .. .. .. ..$ : chr "HuGene-2_0-st.pbs"
  .. .. .. .. .. .. .. ..$ : chr "HuGene-2_0-st.ann"
  .. .. .. .. ..@ data     :'data.frame':       106090 obs. of  8 variables:
  .. .. .. .. .. ..$ UNIT_ID      : int [1:106090] 3742 3743 3744 3745 3747 3748 3749 3750 3751 3752 ...
  .. .. .. .. .. ..$ UnitName     : int [1:106090] 16657437 16657438 16657439 16657441 16657443 16657444 16657446 16657448 16657449 16657451 ...
  .. .. .. .. .. ..$ X_8.mdp_LEVEL: num [1:106090] 2.11 2.55 11.31 4.19 41.9 ...
  .. .. .. .. .. ..$ E.mdp_LEVEL  : num [1:106090] 2.11 1.92 7.27 6.3 22 ...
  .. .. .. .. .. ..$ GG7.mdp_LEVEL: num [1:106090] 1.32 3.63 10.01 2.11 23.65 ...
  .. .. .. .. .. ..$ J.mdp_LEVEL  : num [1:106090] 4.15 2.26 10.02 1.07 10.65 ...
  .. .. .. .. .. ..$ O.mdp_LEVEL  : num [1:106090] 9.77 3.25 15.23 1.43 29.46 ...
  .. .. .. .. .. ..$ T.mdp_LEVEL  : num [1:106090] 14 2.8 6.35 1.74 13.76 ...
  .. .. .. .. ..@ params   : list()
  .. .. .. .. ..@ setname  : chr "PreprocesSet"
  .. .. .. .. ..@ settype  : chr "preprocess"
  .. .. .. .. ..@ rootfile : chr "D:\\GeneST_Analyse\\New4516/rootdata/tmp_exonRMA.root"
  .. .. .. .. ..@ filedir  : chr "D:\\GeneST_Analyse\\New4516/rootdata"
  .. .. .. .. ..@ numtrees : int 6
  .. .. .. .. ..@ treenames:List of 6
  .. .. .. .. .. ..$ : chr "X_8.mdp"
  .. .. .. .. .. ..$ : chr "E.mdp"
  .. .. .. .. .. ..$ : chr "GG7.mdp"
  .. .. .. .. .. ..$ : chr "J.mdp"
  .. .. .. .. .. ..$ : chr "O.mdp"
  .. .. .. .. .. ..$ : chr "T.mdp"
  .. .. ..@ callset  :Formal class 'CallTreeSet' [package "xps"] with 11 slots
  .. .. .. .. ..@ calltype : chr "mas5"
  .. .. .. .. ..@ detcall  :'data.frame':       0 obs. of  0 variables
  .. .. .. .. ..@ scheme   :Formal class 'SchemeTreeSet' [package "xps"] with 12 slots
  .. .. .. .. .. .. ..@ chipname : chr ""
  .. .. .. .. .. .. ..@ chiptype : chr "GeneChip"
  .. .. .. .. .. .. ..@ probeinfo: list()
  .. .. .. .. .. .. ..@ probe    :'data.frame': 0 obs. of  0 variables
  .. .. .. .. .. .. ..@ unitname :'data.frame': 0 obs. of  0 variables
  .. .. .. .. .. .. ..@ mask     :'data.frame': 0 obs. of  0 variables
  .. .. .. .. .. .. ..@ setname  : chr ""
  .. .. .. .. .. .. ..@ settype  : chr ""
  .. .. .. .. .. .. ..@ rootfile : chr "ROOTFile"
  .. .. .. .. .. .. ..@ filedir  : chr "C:/biocbld/bbs-3.2-bioc/tmpdir/Rtmpy2DVDR/R.INSTALLc98a4d4a9/xps"
  .. .. .. .. .. .. ..@ numtrees : num 0
  .. .. .. .. .. .. ..@ treenames: list()
  .. .. .. .. ..@ data     :'data.frame':       0 obs. of  0 variables
  .. .. .. .. ..@ params   : list()
  .. .. .. .. ..@ setname  : chr ""
  .. .. .. .. ..@ settype  : chr ""
  .. .. .. .. ..@ rootfile : chr "ROOTFile"
  .. .. .. .. ..@ filedir  : chr "C:/biocbld/bbs-3.2-bioc/tmpdir/Rtmpy2DVDR/R.INSTALLc98a4d4a9/xps"
  .. .. .. .. ..@ numtrees : num 0
  .. .. .. .. ..@ treenames: list()
  .. .. ..@ scheme   :Formal class 'SchemeTreeSet' [package "xps"] with 12 slots
  .. .. .. .. ..@ chipname : chr "HuGene-2_0-st"
  .. .. .. .. ..@ chiptype : chr "ExonChip"
  .. .. .. .. ..@ probeinfo:List of 8
  .. .. .. .. .. ..$ nrows     : int 1612
  .. .. .. .. .. ..$ ncols     : int 1612
  .. .. .. .. .. ..$ nprobes   : int 1022045
  .. .. .. .. .. ..$ ncontrols : int 23
  .. .. .. .. .. ..$ ngenes    : int 48204
  .. .. .. .. .. ..$ nunits    : int 51991
  .. .. .. .. .. ..$ nprobesets: int 352859
  .. .. .. .. .. ..$ naffx     : int 167
  .. .. .. .. ..@ probe    :'data.frame':       0 obs. of  0 variables
  .. .. .. .. ..@ unitname :'data.frame':       0 obs. of  0 variables
  .. .. .. .. ..@ mask     :'data.frame':       0 obs. of  0 variables
  .. .. .. .. ..@ setname  : chr "HuGene-2_0-st"
  .. .. .. .. ..@ settype  : chr "scheme"
  .. .. .. .. ..@ rootfile : chr "D:\\GeneST_Analyse\\New4516/schemes/Scheme.root"
  .. .. .. .. ..@ filedir  : chr "D:\\GeneST_Analyse\\New4516/schemes"
  .. .. .. .. ..@ numtrees : int 9
  .. .. .. .. ..@ treenames:List of 9
  .. .. .. .. .. ..$ : chr "HuGene-2_0-st.cxy"
  .. .. .. .. .. ..$ : chr "HuGene-2_0-st.anx"
  .. .. .. .. .. ..$ : chr "HuGene-2_0-st.anp"
  .. .. .. .. .. ..$ : chr "HuGene-2_0-st.scm"
  .. .. .. .. .. ..$ : chr "HuGene-2_0-st.idx"
  .. .. .. .. .. ..$ : chr "HuGene-2_0-st.prb"
  .. .. .. .. .. ..$ : chr "HuGene-2_0-st.exn"
  .. .. .. .. .. ..$ : chr "HuGene-2_0-st.pbs"
  .. .. .. .. .. ..$ : chr "HuGene-2_0-st.ann"
  .. .. ..@ data     :'data.frame':     345497 obs. of  3 variables:
  .. .. .. ..$ UNIT_ID : int [1:345497] 3742 3743 3744 3745 3746 3747 3748 3749 3750 3751 ...
  .. .. .. ..$ UnitName: int [1:345497] 16657437 16657438 16657439 16657441 16657442 16657443 16657444 16657446 16657448 16657449 ...
  .. .. .. ..$ FLAG    : int [1:345497] 0 0 0 0 0 0 0 0 0 0 ...
  .. .. ..@ params   : list()
  .. .. ..@ setname  : chr "UniFilterSet"
  .. .. ..@ settype  : chr "unifilter"
  .. .. ..@ rootfile : chr "D:\\GeneST_Analyse\\New4516/rootdata/tmp_exonUnifilter_ufr.root"
  .. .. ..@ filedir  : chr "D:\\GeneST_Analyse\\New4516/rootdata"
  .. .. ..@ numtrees : int 1
  .. .. ..@ treenames:List of 1
  .. .. .. ..$ : chr "UniTest.ufr"
  ..@ scheme   :Formal class 'SchemeTreeSet' [package "xps"] with 12 slots
  .. .. ..@ chipname : chr "HuGene-2_0-st"
  .. .. ..@ chiptype : chr "ExonChip"
  .. .. ..@ probeinfo:List of 8
  .. .. .. ..$ nrows     : int 1612
  .. .. .. ..$ ncols     : int 1612
  .. .. .. ..$ nprobes   : int 1022045
  .. .. .. ..$ ncontrols : int 23
  .. .. .. ..$ ngenes    : int 48204
  .. .. .. ..$ nunits    : int 51991
  .. .. .. ..$ nprobesets: int 352859
  .. .. .. ..$ naffx     : int 167
  .. .. ..@ probe    :'data.frame':     0 obs. of  0 variables
  .. .. ..@ unitname :'data.frame':     0 obs. of  0 variables
  .. .. ..@ mask     :'data.frame':     0 obs. of  0 variables
  .. .. ..@ setname  : chr "HuGene-2_0-st"
  .. .. ..@ settype  : chr "scheme"
  .. .. ..@ rootfile : chr "D:\\GeneST_Analyse\\New4516/schemes/Scheme.root"
  .. .. ..@ filedir  : chr "D:\\GeneST_Analyse\\New4516/schemes"
  .. .. ..@ numtrees : int 9
  .. .. ..@ treenames:List of 9
  .. .. .. ..$ : chr "HuGene-2_0-st.cxy"
  .. .. .. ..$ : chr "HuGene-2_0-st.anx"
  .. .. .. ..$ : chr "HuGene-2_0-st.anp"
  .. .. .. ..$ : chr "HuGene-2_0-st.scm"
  .. .. .. ..$ : chr "HuGene-2_0-st.idx"
  .. .. .. ..$ : chr "HuGene-2_0-st.prb"
  .. .. .. ..$ : chr "HuGene-2_0-st.exn"
  .. .. .. ..$ : chr "HuGene-2_0-st.pbs"
  .. .. .. ..$ : chr "HuGene-2_0-st.ann"
  ..@ data     :'data.frame':   345497 obs. of  10 variables:
  .. ..$ UNIT_ID        : int [1:345497] 3742 3743 3744 3745 3746 3747 3748 3749 3750 3751 ...
  .. ..$ UnitName       : int [1:345497] 16657437 16657438 16657439 16657441 16657442 16657443 16657444 16657446 16657448 16657449 ...
  .. ..$ Statistics     : num [1:345497] -2.603 -0.117 -0.353 2.272 -2.333 ...
  .. ..$ Mean1          : num [1:345497] 1.85 2.7 9.53 4.2 2.11 ...
  .. ..$ Mean2          : num [1:345497] 9.3 2.77 10.53 1.41 3.38 ...
  .. ..$ StandardError  : num [1:345497] 2.865 0.575 2.836 1.227 0.542 ...
  .. ..$ DegreeOfFreedom: int [1:345497] 4 4 4 4 4 4 4 4 4 4 ...
  .. ..$ P-Value        : num [1:345497] 0.0599 0.9125 0.742 0.0855 0.08 ...
  .. ..$ P-Adjusted     : num [1:345497] 0.0599 0.9125 0.742 0.0855 0.08 ...
  .. ..$ FoldChange     : num [1:345497] 5.038 1.025 1.105 0.337 1.599 ...
  ..@ params   : list()
  ..@ setname  : chr "UniFilterSet"
  ..@ settype  : chr "UnivariateAnalysis"
  ..@ rootfile : chr "D:\\GeneST_Analyse\\New4516/rootdata/tmp_exonUnifilter_ufr.root"
  ..@ filedir  : chr "D:\\GeneST_Analyse\\New4516/rootdata"
  ..@ numtrees : int 1
  ..@ treenames:List of 1
  .. ..$ : chr "UniTest.stt"

How come there are extra probesets after differential analysis?

head(tmp)
         UNIT_ID Statistics    Mean1    Mean2 StandardError
16657452    3753    2.83827  7.25055  3.29028       1.39531
16657519    3802   -3.04815  6.39265 19.06580       4.15766
16657589    3857   -2.86559  4.47849 10.86110       2.22734
16657604    3867   -3.68341 22.02750 37.22920       4.12707
16657629    3886   -3.18077 25.43610 58.89590      10.51940
16657644    3896   -3.18920  3.34795 15.95470       3.95297
         DegreeOfFreedom   P-Value P-Adjusted FoldChange
16657452               4 0.0469499  0.0469499   0.453797
16657519               4 0.0380969  0.0380969   2.982460
16657589               4 0.0456711  0.0456711   2.425180
16657604               4 0.0211407  0.0211407   1.690120
16657629               4 0.0335105  0.0335105   2.315440
16657644               4 0.0332417  0.0332417   4.765530

None of the UnitNames present in "tmp" are from subset3.rma  And why it is giving other Unitnames and some extra UnitNames also? @cstrato

 

 

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Entering edit mode
cstrato ★ 3.9k
@cstrato-908
Last seen 6.1 years ago
Austria

Since the replies are becoming unreadable, I will create a new answer. Please reply to this answer.

Please note that the replacement function: 'exprs(object, treenames = NULL) <- value'
is introduced to allow to replace slot 'data' containing the expression data for all trees with 
expression data for a subset of trees, see help '?exprs'
This means that the number of rows and the rownames must be identical to the ones of the original
dataframe 'data'.

Regards,
Christian
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Entering edit mode

the "value" have identical columns but not identical rows to the original, cz as i already said you that there are multiple probesets for each transcript so, I calculated mean and then took the best probesets for differential analysis. I guess its hard to use unifilter now for differential analysis. Could you please recommend any other way for differential analysis? I can go for limma but it calculates FC in a different way from unifilter does. So, I'm not interested in limma at all.

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Entering edit mode

Since you have two groups, group=c("GrpA","GrpA","GrpA","GrpB","GrpB","GrpB"), and unifliter() 
uses the t-test by default, simply apply function t.test() of basic R to your two groups.
However, I still suggest to use limma.

Regards,
Christian

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