Dear All,

Tried rerunning an R file that I made about a year ago to analyse TLDA array cards. However, when arriving at determining differentially expressed miRNAs I get an error where this returned a perfect set of differentially expressed miRNAs a year ago.

sampleinfo<-read.delim("sampleinfo.txt")
design <- model.matrix(~0 + sampleinfo$Function)
colnames(design) <- c("EAD", "IF")
design
contrasts <- makeContrasts(EAD - IF,levels = design)
colnames(contrasts) <- c("EAD - IF")
contrasts
gnorm.raw.cat.Filt2<-gnorm.raw.cat.Filt[order(featureNames(gnorm.raw.cat.Filt))]
qDE.gnorm.raw.cat.Filt2.limma <- limmaCtData(gnorm.raw.cat.Filt2,design=design, contrasts=contrasts,  stringent = FALSE, sort=TRUE)

Error in data.frame(..., check.names = FALSE) : 
  arguments imply differing number of rows: 192, 0

I checked the object gnorm.raw.cat.Filt2 but 

 @ featureData

      @ data indicates a 'data.frame' of 192 obs. of 1 variable:

Being a beginner in R it took me a lot of time to get this package to work.

> sessionInfo()
R version 3.4.0 (2017-04-21)
Platform: x86_64-w64-mingw32/x64 (64-bit)
Running under: Windows 7 x64 (build 7601) Service Pack 1

Matrix products: default

locale:
[1] LC_COLLATE=English_United States.1252  LC_CTYPE=English_United States.1252    LC_MONETARY=English_United States.1252
[4] LC_NUMERIC=C                           LC_TIME=English_United States.1252    

attached base packages:
[1] parallel  stats     graphics  grDevices utils     datasets  methods   base     

other attached packages:
[1] HTqPCR_1.30.0       limma_3.32.10       RColorBrewer_1.1-2  Biobase_2.36.2      BiocGenerics_0.22.1

loaded via a namespace (and not attached):
 [1] gtools_3.5.0          bitops_1.0-6          affy_1.54.0           stats4_3.4.0          KernSmooth_2.23-15    BiocInstaller_1.26.1 
 [7] gplots_3.0.1          zlibbioc_1.22.0       gdata_2.18.0          affyio_1.46.0         preprocessCore_1.38.1 tools_3.4.0          
[13] compiler_3.4.0        caTools_1.17.1 

Dear h.roest,

looking at your code, I see that gnorm.raw.cat.Filt being used without ever being read from a data file

or otherwise created.  Is it possible that you're running your R script line by line and accidentally

skipped one or more lines?  Just a guess.

Cheers,

 - axel

Dear Axel,

Thanks for your comment. Unfortunately, the "gnorm.raw.cat.Filt" object was created by filtering out the targets that were more then 4 out of 8 Undetermined. It looks that the problem actually starts here. limma of the unfiltered HTqPCR object can nicely pass the differential expression using limmaCtData. Remarkably, mutest and ttest work fine on the gnorm.raw.cat.Filt object. Maybe you have some more suggestions on how to solve this problem?

Cheers,

Henk