[BioC] Reading in data with HTqPCR
Heidi Dvinge
heidi at ebi.ac.uk
Sun Jun 6 12:29:35 CEST 2010
> Dear sir,
>
Hello Deepak,
again, please keep all discussion to the list! I.e. reply to all
(including bioconductor at stat.math.ethz.ch) and not just the person who
answered your question.
>
> 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)
>
Glad to hear that it worked out.
>
> 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.
>
Why not? DeltaCt is just one type of normalisation, out of 4 currently
implemented in HTqPCR. As such, you can select just a single
normalisation, or none at all depending on how much you trust your initial
data, and proceed with that.
That being said, in your example below you use the command:
>> normalizeCtData(raw.cat, norm = "deltaCt", deltaCt.genes = c("Gene11"),
> scale.rank.samples, Ct.max = 38, verbose = TRUE)
I.e. you normalise everything to the expression of a feature on your card
called "Gene11". Do you have a gene by that name in your
featureNames(raw.cat)? If not, then you're trying to normalise against
nothing... Maybe you want deltaCt.genes="18sRNA"?
HTH
\Heidi
> 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 at 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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