Hi,
i'm trying to read a tab delimited file with read.qPCR in package ReadqPCR to use the data with NormqPCR to apply geNorm method to obtain the best housekeeping genes in my experiment.
The data were obtained with Absolute Quantification (PCR kit supplier requisite) in a 7900HT qPCR.
I've tried with read.taqman, but I have no Summary info in my SDS exported file, so I'm trying the more generic read.qPCR function, but I'm stuck.
The experiment were 4 384 plates, with 4 samples in each plate (format 96 x 4). I formated my text file as if each 384 plate were indeed 4 96 plates. Detector column was created for the first sample, and copy-pasted for all the remaining 15, so the detector names are the same. I've checked for spaces in the detector names or between the name and the next column (Cq).
There are no replicates but for genomic, positive and negative controls (the last 10 wells for every sample). I've ordered my input file by sample name, and I've even deleted the two firt optional columns just in case.
I can't figure out what I'm missing. Could you please give me advise for this?
This is my code and output:
> library(Biobase)
Loading required package: BiocGenerics
Loading required package: parallel
Attaching package: ‘BiocGenerics’
The following objects are masked from ‘package:parallel’:
clusterApply, clusterApplyLB, clusterCall, clusterEvalQ, clusterExport, clusterMap, parApply, parCapply, parLapply,
parLapplyLB, parRapply, parSapply, parSapplyLB
The following objects are masked from ‘package:stats’:
IQR, mad, xtabs
The following objects are masked from ‘package:base’:
anyDuplicated, append, as.data.frame, as.vector, cbind, colnames, do.call, duplicated, eval, evalq, Filter, Find,
get, grep, grepl, intersect, is.unsorted, lapply, lengths, Map, mapply, match, mget, order, paste, pmax, pmax.int,
pmin, pmin.int, Position, rank, rbind, Reduce, rownames, sapply, setdiff, sort, table, tapply, union, unique,
unlist, unsplit
Welcome to Bioconductor
Vignettes contain introductory material; view with 'browseVignettes()'. To cite Bioconductor, see
'citation("Biobase")', and for packages 'citation("pkgname")'.
> library(lattice)
> library(ReadqPCR)
Loading required package: affy
> library(NormqPCR)
Loading required package: RColorBrewer
Loading required package: qpcR
Loading required package: MASS
Loading required package: minpack.lm
Loading required package: rgl
Loading required package: robustbase
Attaching package: ‘robustbase’
The following object is masked from ‘package:Biobase’:
rowMedians
Loading required package: Matrix
> setwd("C:/Users/Transcriptomics/Desktop/data")
> path <- getwd()
> taqman <- file.path(path, "plates_read2.txt")
> taqman
[1] "C:/Users/Transcriptomics/Desktop/data/plates_read2.txt"
> content <- read.table("plates_read2.txt", header=TRUE, sep="\t")
> content[c(1:3, 98:100, 194:196),]
Sample Detector Cq
1 C1 AHNAK 22.64534
2 C1 AKT1 24.03385
3 C1 BMP1 24.89237
98 C2 AKT1 24.25720
99 C2 BMP1 25.75276
100 C2 BMP2 25.57264
194 C3 AKT1 26.31538
195 C3 BMP1 27.33528
196 C3 BMP2 29.18772
> qPCRBatch.taqman <- read.qPCR(taqman)
Error in .read.qPCR(filename, verbose) :
File incorrect, make sure that detectors are the same for all samples
> sessionInfo()
R version 3.2.3 (2015-12-10)
Platform: x86_64-w64-mingw32/x64 (64-bit)
Running under: Windows 7 x64 (build 7601) Service Pack 1
locale:
[1] LC_COLLATE=Spanish_Spain.1252 LC_CTYPE=Spanish_Spain.1252 LC_MONETARY=Spanish_Spain.1252 LC_NUMERIC=C
[5] LC_TIME=Spanish_Spain.1252
attached base packages:
[1] parallel stats graphics grDevices utils datasets methods base
other attached packages:
[1] NormqPCR_1.16.0 qpcR_1.4-0 Matrix_1.2-3 robustbase_0.92-5 rgl_0.95.1441 minpack.lm_1.2-0
[7] MASS_7.3-45 RColorBrewer_1.1-2 ReadqPCR_1.16.0 affy_1.48.0 lattice_0.20-33 Biobase_2.30.0
[13] BiocGenerics_0.16.1
loaded via a namespace (and not attached):
[1] grid_3.2.3 BiocInstaller_1.20.1 zlibbioc_1.16.0 affyio_1.40.0 preprocessCore_1.32.0
[6] tools_3.2.3 DEoptimR_1.0-4
Do you have any values that appear more times in the Detector column for the same sample than others? That seems to be what actually triggers that error message. It isn't actually checking anything between the samples.
For example, this will throw that error because AKT1 appears twice, but you only have the other detectors represented once, even though there's only one sample group.
If that doesn't help you track it down, maybe make the whole plates_read2.txt file available somewhere.
Hi,
no, there are exactly the same number of samples for each detector; there are 16 samples, and all of them have the same "kit" of detectors. It was my mistake in selecting one of the data to show in the post that make it seem as being represented in different number.
The original plates are 384 wells (4 plates in total), with 4 samples each. A1, A2, B1 and B2 wells have the same detector, and so on. There are no replicates, and even I have erased from the original data, the genomic, positive, and negative controls, as they were the only ones spotted in triplicate.
Best,
Elena
I'm having the same issue as you, Elena. It seems that this package cannot cope with larger numbers of genes and samples since it worked fine with a small subset of my data but as soon as I add more genes and samples it comes up with the "make sure that detectors are the same for all samples" when they are! Would welcome any suggestions. In the meantime, I'll do this manually.