Dear sir,





The previous problem was solved that was due to the Ct min. Because as
default ct min was 10 but in my card i used 18sRNA which had ct below 10
along with that the quantile i changed to NULL from 0.8 because i didn’t had
replicates of samples i change the whole command as following then it worked

>raw.cat <- setCategory(raw, Ct.max = 38, Ct.min = 5, replicates = TRUE,
quantile = NULL, groups, flag = TRUE, flag.out = "Failed", verbose = TRUE,
plot = FALSE)



 and two type of normalization worked but the norm.rank and deltact type
normalization didn’t worked details are given below. Without delta ct
normalization i not able to further analysis.

 deepak



> nr.norm <- normalizeCtData(raw.cat, norm="norm.rank")

Normalizing Ct values

        Using rank invariant genes:

        Sample4: 16 rank invariant genes

        Sample67: 19 rank invariant genes

        Sample80: 21 rank invariant genes

        Sample44: 21 rank invariant genes

        Sample76: 18 rank invariant genes

        Sample94: 22 rank invariant genes

        Sample17: 14 rank invariant genes

        Sample37: 2 rank invariant genes

        Sample32: 14 rank invariant genes

        Sample33: 12 rank invariant genes

        Sample34: 14 rank invariant genes

        Sample25: 11 rank invariant genes

        Sample26: 10 rank invariant genes

Warning message:

In approx(ri.sub$n.curve$y, ri.sub$n.curve$x, xout = data[, i],  :

  collapsing to unique 'x' values

> traceback()

No traceback available





> normalizeCtData(raw.cat, norm = "deltaCt", deltaCt.genes = c("Gene11"),
scale.rank.samples,  Ct.max = 38, verbose = TRUE)

Calculating deltaCt values

        Using control gene(s): Gene11

        Card 1: Mean=NaN        Stdev=NA

        Card 2: Mean=NaN        Stdev=NA

        Card 3: Mean=NaN        Stdev=NA

        Card 4: Mean=NaN        Stdev=NA

        Card 5: Mean=NaN        Stdev=NA

        Card 6: Mean=NaN        Stdev=NA

        Card 7: Mean=NaN        Stdev=NA

        Card 8: Mean=NaN        Stdev=NA

        Card 9: Mean=NaN        Stdev=NA







        Card 92:        Mean=NaN        Stdev=NA

        Card 93:        Mean=NaN        Stdev=NA

        Card 94:        Mean=NaN        Stdev=NA

        Card 95:        Mean=NaN        Stdev=NA

        Card 96:        Mean=NaN        Stdev=NA

        Card 97:        Mean=NaN        Stdev=NA

        Card 98:        Mean=NaN        Stdev=NA

        Card 99:        Mean=NaN        Stdev=NA

An object of class "qPCRset"

Size:  48 features, 99 samples

Feature types:           Endogenous Control, Target

Feature names:           CCNF RBL2 CCND1 ...

Feature classes:

Feature categories:      OK, Undetermined

Sample names:            Sample4 Sample67 Sample80 ...

> traceback()

No traceback available

> sessionInfo()

R version 2.11.0 (2010-04-22)

x86_64-pc-mingw32



locale:

[1] LC_COLLATE=English_India.1252  LC_CTYPE=English_India.1252
LC_MONETARY=English_India.1252
LC_NUMERIC=C                   LC_TIME=English_India.1252



attached base packages:

[1] stats     graphics  grDevices utils     datasets  methods   base



other attached packages:

[1] HTqPCR_1.2.0       limma_3.4.1        RColorBrewer_1.0-2 Biobase_2.8.0




loaded via a namespace (and not attached):

[1] affy_1.26.1           affyio_1.16.0         gdata_2.8.0
gplots_2.7.4          gtools_2.6.2          preprocessCore_1.10.0

>







deepak


On 6/4/10, Heidi Dvinge <heidi@ebi.ac.uk> wrote:
>
>  Hello Deepak,
>
>
> please keep conversations on the BioC email list. As I mentioned in my
> previous email to you, the BioC email archive is a valuable source of
> information in addition to vignettes and the default "?" help pages.
>
> Dear sir,
> The previous problem was solved and that occurred due one of sample had
> only 47 gene data instead of 48.
>
>
>
> Glad to hear that has worked out.
>
> >when i give  raw.cat <- setCategory(raw, groups=files$Treatment,
> quantile=0.8) for all 99 sample Unreliable, Undetermined, OK values came all
> 99 sample showed 48 gene when we add Unreliable, Undetermined, OK but in
> error it is shown as *missing values* are not allowed in subscripted
> assignments of data frames.
>
> deepak
>
>
>
> > path <- "F:/HTqPCR/TLDA"
>
> > exPath <- "F:/HTqPCR/TLDA"
>
> > exFiles <- read.delim(file.path(exPath, "files.txt"))
>
> > raw <- readCtData(files=exFiles$File[c(1:99)], path=exPath, n.features =
> 48, flag = 4, feature = 6, type = 7, position = 3, Ct = 8, header = FALSE,
> SDS = FALSE, n.data = 1, na.value = 40)
>
> > raw
>
> An object of class "qPCRset"
>
> Size:  48 features, 99 samples
>
> Feature types:           Endogenous Control, Target
>
> Feature names:           CCNF RBL2 CCND1 ...
>
> Feature classes:
>
> Feature categories:      OK, Undetermined
>
> Sample names:            Sample4 Sample67 Sample80 ...
>
> > path <- "F:/HTqPCR/TLDA"
>
> > files <- read.delim(file.path(path, "files.txt"))
>
> > g <- featureNames(raw)[1:48]
>
> > plotCtOverview(raw, genes=g, xlim=c(0,150), groups=files$Treatment,
> conf.int=TRUE,
>
> + ylim=c(0,55))
>
> > plotCtOverview(raw, genes=g, xlim=c(0,150), groups=files$Treatment,
>
> + calibrator="Normal")
>
> > plotCtCard(raw, col.range=c(10,35), well.size=2.6)
>
> > featureClass(raw) <- factor(rep(c("Cyclin", "Cyclin_Inhibitor"),
> times=c(10,38)))
>
> > plotCtCard(raw, plot="class", well.size=2.6)
>
> > plotCtCard(raw, col.range=c(10,35), well.size=2.6)
>
>
>
>
>
> > raw.cat <- setCategory(raw, groups=files$Treatment, quantile=0.8)
>
> Categories after Ct.max and Ct.min filtering:
>
>
>
> Error in `[<-.data.frame`(`*tmp*`, index, value = "Unreliable") :
>
>   missing values are not allowed in subscripted assignments of data frames
>
>
>
> I have seen something like this once before (it has been fixed in HTqPCR
> devel), but that was after using the rbind and cbind functions so I'm not
> sure what's going on here. What does:
>
>
> class(featureCategory(raw))
>  class(featureCategory(raw)[,1])
>
>
> say? If the second says "factor" instead of "character", then try typing:
>
>
> featureCategory(raw) <- data.frame(apply(featureCategory(raw),
> 2, as.character), stringsAsFactors=FALSE)
>
>
> Also, what's currently in your featureCategory? Try saying:
>
>
> head(featureCategory(raw))
>
>
> HTH
> \Heidi
>
>
>
>  > traceback()
>
> 4: stop("missing values are not allowed in subscripted assignments of data
> frames")
>
> 3: `[<-.data.frame`(`*tmp*`, index, value = "Unreliable")
>
> 2: `[<-`(`*tmp*`, index, value = "Unreliable")
>
> 1: setCategory(raw, groups = files$Treatment, quantile = 0.8)
>
> > sessionInfo()
>
> R version 2.11.0 (2010-04-22)
>
> x86_64-pc-mingw32
>
>
>
> locale:
>
> [1] LC_COLLATE=English_India.1252  LC_CTYPE=English_India.1252
>
> [3] LC_MONETARY=English_India.1252 LC_NUMERIC=C
>
> [5] LC_TIME=English_India.1252
>
>
>
> attached base packages:
>
> [1] stats     graphics  grDevices utils     datasets  methods   base
>
>
>
> other attached packages:
>
> [1] HTqPCR_1.2.0       limma_3.4.1        RColorBrewer_1.0-2 Biobase_2.8.0
>
>
>
>
> loaded via a namespace (and not attached):
>
> [1] affy_1.26.1           affyio_1.16.0         gdata_2.8.0
>
> [4] gplots_2.7.4          gtools_2.6.2          preprocessCore_1.10.0
>
>
>
>
> --
> Deepak Roshan V G
> Laboratory Of Cell Cycle Regulation  &   Molecular Oncology
> Division of Cancer Research
> Regional Cancer Centre
> Thiruvananthapuram
> Kerala, India 695 011
>
>
>
>



-- 
Deepak Roshan V G
Laboratory Of Cell Cycle Regulation  &   Molecular Oncology
Division of Cancer Research
Regional Cancer Centre
Thiruvananthapuram
Kerala, India 695 011

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