[BioC] Problems creating a qPCRset object in "HTqPCR"

[Peter Davidsen]

Dear List,

I'm trying to use the "HTqPCR" BioC package to analyze "SDS-format"
qPCR data (ran 384-well plates on an ABI 7900HT).
I have 20 different txt-files (in SDS-format) as I quantified 20
different miRNAs (thus, only one GOI per plate/file) - on each plate I
have 33 samples in triplicate.

Example of how my txt-files are organized:

SDS 2.3	AQ Results	1.0
Filename	05.08.2010_miR-208b_Ceramide_1-33
PlateID	
Assay Type	Absolute Quantification
Run DateTime	05/08/10 14:28:11
Operator	
ThermalCycleParams	

Sample Information

Well	Sample Name	Detector Name	Reporter	Task	Ct....
82	D10	miR-208b	FAM	Unknown	31.154957																				
83	D11	miR-208b	FAM	Unknown	30.56075																			
84	D12	miR-208b	FAM	Unknown	30.617414																			
85	D13	miR-208b	FAM	Unknown	29.638447																			
86	D14	miR-208b	FAM	Unknown	29.873674																			
87	D15	miR-208b	FAM	Unknown	29.974564																			
88	D16	miR-208b	FAM	Unknown	32.324512																	
89	D17	miR-208b	FAM	Unknown	31.65119																		
90	D18	miR-208b	FAM	Unknown	32.567085																		
91	D19	miR-208b	FAM	Unknown	30.977701																		
92	D20	miR-208b	FAM	Unknown	31.459805	



The command I run is:
# Load HTqPCR package
library("HTqPCR")

# Set working dir
path <- setwd('C:/Documents and
Settings/RH35363/Dokumenter/Dropbox/Ceramide Study/SDS-files')

exFiles <- read.delim(file.path(path, "files.txt")) # "files.txt"
contains a list with the file names of my 20 txt-files

raw <- readCtData(files=exFiles$File, path=path, n.features = 99, flag
= NULL, feature = 3, type = 5, position = 2,
Ct = 6, n.data = 33, na.value = 40, header=T, SDS = FALSE, skip = 10,
sep='\t')  # Since my headers are apparently different/in a different
order, I have set these parameters accordingly. I've set SDS=FALSE as
my SDS software generated txt-files lack a "#" in the first column.

And the error I get is:
Error in `[<-.data.frame`(`*tmp*`, undeter, value = "Undetermined") :
  only logical matrix subscripts are allowed in replacement
In addition: Warning messages:
1: In readCtData(files = "05.08.2010_miR-208b_Ceramide_1-33.txt", path
= NULL,  :
  99 gene names (rows) expected, got 1132
2: In matrix(sample[, Ct], ncol = n.data[i]) :
  data length [116] is not a sub-multiple or multiple of the number of
columns [33]

With the traceback information as follows:
> traceback()
4: stop("only logical matrix subscripts are allowed in replacement")
3: `[<-.data.frame`(`*tmp*`, undeter, value = "Undetermined")
2: `[<-`(`*tmp*`, undeter, value = "Undetermined")
1: readCtData(files = exFiles$File, path = path, n.features = 99,
       flag = NULL, feature = 3, type = 5, position = 2, Ct = 6,
       n.data = 33, na.value = 40, header = T, SDS = FALSE, skip = 10,
       sep = "\t")


I tried to change the "n.data" parameter from 33 to 1, this removed the error:
raw <- readCtData(files=exFiles$File, path=path, n.features = 99, flag
= NULL, feature = 3, type = 5, position = 2,
+ Ct = 6, n.data = 1, header=T, SDS = FALSE, skip = 10, sep='\t')
Warning message:
In readCtData(files = exFiles$File, path = path, n.features = 93,  :
  99 gene names (rows) expected, got 1132

Thus, it seems to me like I'm loading my data in the wrong way. Any
thoughts/suggestions on how to solve this problem are welcomed!

> sessionInfo()
R version 2.12.2 (2011-02-25)
Platform: i386-pc-mingw32/i386 (32-bit)

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

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

other attached packages:
[1] HTqPCR_1.4.0       limma_3.6.9        RColorBrewer_1.0-2 Biobase_2.10.0

loaded via a namespace (and not attached):
[1] affy_1.28.1           affyio_1.18.0         gdata_2.8.2
[4] gplots_2.8.0          gtools_2.6.2          preprocessCore_1.12.0


Kind regards,
Peter