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Guest User ★ 12k@guest-user-4897
Last seen 8.4 years ago
Dear James, Thanks for your time and help with ReadqPCR and NormqPCR. I have worked a bit more with this NormqPCR; Very useful package, especially, for normalizing raw Ct data, determining housekeeping genes, etc. Thus far, I have been successful in working through the vignette. Although, I have one last inquiry. In order to confirm where the error is coming from, I have deduced my input file containing the raw Cq values to 2 conditions, 8 detectors, one technical replication. > qPCRBatch=read.qPCRqPCRBatch.lv) Warning message: In read.qPCRqPCRBatch.lv) : Incompatible phenoData object. Created a new one using sample name data derived from raw data. > exprs(qPCRBatch) caseA control actin 20.89 20.91 lox22 21.36 20.50 lox23 20.93 21.00 lox34 20.89 20.80 lox61 19.50 19.50 lox62 21.00 25.00 lox69 19.30 15.00 lox99 21.10 28.00 > contM=cbind(c(1,0), c(0,1)) > colnames(contM)=c("interest", "no interest") > rownames(contM)=sampleNamesqPCRBatch.lv) > contM interest no interest caseA 1 0 control 0 1 > hkgs="actin" > ddCq.lv=deltaDeltaCqqPCRBatch.lv, maxNACase=1, maxNAControl=1, hkgs=hkgs, contrastM=contM, case="interest", control="no interest", statCalc="arith", hkgCalc="arith") Error in caseM[hkgs, ] : incorrect number of dimensions Apart from the vignette, I didn't think I would need to identify hkgs in each sample?? What is odd is that I can complete the deltaCq function, > qPCRBatch.dcq=deltaCqqPCRBatch.lv, hkgs=hkgs, calc="arith") > exprs(qPCRBatch.dcq) caseA control actin 0.00 0.00 lox22 0.47 -0.41 lox23 0.04 0.09 lox34 0.00 -0.11 lox61 -1.39 -1.41 lox62 0.11 4.09 lox69 -1.59 -5.91 lox99 0.21 7.09 , but not the deltaDeltaCq function. This says to me that there is something wrong with my "case" vs. "control" comparison in the contrast matrix? However, like the vignette, I have my hkgs genes in both samples, as required by the same detectors for all samples requisite(which makes sense). Anyhow, it's been great to work with NormqPCR. Hopefully, I can complete it by getting the deltaDeltaCq output. If you have a chance, can you please take a look at the scripts and correct me where I'm wrong. Respectfully, Franklin -- output of sessionInfo(): > sessionInfo() R version 2.15.1 (2012-06-22) Platform: x86_64-pc-mingw32/x64 (64-bit) locale:  LC_COLLATE=English_United States.1252 LC_CTYPE=English_United States.1252 LC_MONETARY=English_United States.1252  LC_NUMERIC=C LC_TIME=English_United States.1252 attached base packages:  stats graphics grDevices utils datasets methods base other attached packages:  limma_3.14.0 NormqPCR_1.4.0 RColorBrewer_1.0-5 ReadqPCR_1.4.0 affy_1.36.1 Biobase_2.18.0 BiocGenerics_0.4.0 loaded via a namespace (and not attached):  affyio_1.26.0 BiocInstaller_1.8.3 preprocessCore_1.20.0 tools_2.15.1 zlibbioc_1.4.0 -- Sent via the guest posting facility at bioconductor.org.