Hi Christian,

For RNA degradation plot I am getting an error. Please check the following:

load existing ROOT scheme file and ROOT data file

scheme.exon <- root.scheme(paste(scmdir,"Scheme_HuGene20stv1_na35.root",sep="/"))
data.exon <- root.data(scheme.exon, paste("D:/GeneST_Analyse/CELFiles/rootdata/tmp_data_exon_cel.root",sep="/"))

qualification - rlm

rlm.all <- rmaPLM(data.exon, "tmp_exonRLMall", filedir=getwd(), tmpdir="", qualopt="all", option="transcript", exonlevel="affx+core", add.data=FALSE)

(or)

rlm.all <- fitRLM(data.exon,"tmp_exonRLM", qualopt="all", option="transcript", exonlevel="affx+core", verbose=FALSE)

And I get the following error:

Creating new temporary file <D:/GeneST_Analyse/CELFiles/tmp_exonRLMall.root>...
Opening file <D:/GeneST_Analyse/CELFiles/schemes/Scheme_HuGene20stv1_na35.root>
in <READ> mode...
Opening file <D:/GeneST_Analyse/CELFiles/rootdata/tmp_data_exon_cel.root> in <RE
AD> mode...
Preprocessing data using method <preprocess>...
   Calculating quality control for <raw> data trees  ...
      setting selector mask for typepm <9276>
      setting selector mask for typepm <9276>
      calculating quality controls for <44796> of <51991> units...Finished.
      calculating border elements for <X_8__HuGene_2_0_st__raw.brd>...
      center of intensity:
         positive border elements (x,y) = <0.301687, 0.0508869>.
         negative border elements (x,y) = <-0.228422, -0.0683751>.
      calculating border elements for <E__HuGene_2_0_st__raw.brd>...
      center of intensity:
         positive border elements (x,y) = <0.0752159, 0.194383>.
         negative border elements (x,y) = <-0.159563, -0.0453415>.
      calculating border elements for <GG7__HuGene_2_0_st__raw.brd>...
      center of intensity:
         positive border elements (x,y) = <0.481647, -0.0256652>.
         negative border elements (x,y) = <-0.196879, -0.0731834>.
      calculating border elements for <J__HuGene_2_0_st__raw.brd>...
      center of intensity:
         positive border elements (x,y) = <0.112615, 0.00776396>.
         negative border elements (x,y) = <-0.1127, -0.0283493>.
      calculating border elements for <O__HuGene_2_0_st__raw.brd>...
      center of intensity:
         positive border elements (x,y) = <0.119527, 0.141888>.
         negative border elements (x,y) = <-0.124044, -0.0194095>.
      calculating border elements for <T__HuGene_2_0_st__raw.brd>...
      center of intensity:
         positive border elements (x,y) = <0.219297, -0.114559>.
         negative border elements (x,y) = <-0.144787, 0.0191627>.
   Background correcting raw data...
      calculating background for <X_8__HuGene_2_0_st_.cel>...
Error: Number of PMs or MMs is zero.
An error has occured: Need to abort current process.
Error in .local(object, ...) : error in rwrapper function 'Preprocess'

Please help me out. Thank you !

Dear Venkatesh,

Please note that whole genome/exon arrays do only have PM probes and no MM probes.
For this reason you cannot use the default parameter 'background = "pmonly"', but
you need to use 'background = "antigenomic"', see help: ?fitRLM

Thus you need to use:

rlm.all <- fitRLM(data.exon, "tmp_exonRLM", background="antigenomic", qualopt="all", option="transcript", exonlevel="affx+core")

Regards,
Christian

Dear Christian,

Please click the following URL for RNA degradation plot.

http://i.imgur.com/IacdSwX.png

Don't know why it looks like that. I got the same plot with other data too.

Could you please tell me where I went wrong.

Thank you !

Dear Venkatesh,

RNA degradation plots are used for Affymetrix IVT-arrays to test the quality of the sample mRNA
used for conversion into cDNA. For IVT-arrays Affymetrix used T7-oligo(dT)-Primers, thus package
'affy' has implemented function 'plotAffyRNAdeg()' to show the quality of the mRNA used, see §4.3:
http://bioconductor.org/packages/3.2/bioc/vignettes/affy/inst/doc/affy.pdf

For a detailed explanation you can also see:
http://www.math.usu.edu/~jrstevens/stat5570/Bowles.pdf

With the introduction of whole genome/exon arrays Affymetrix has switched from oligo(dT) primers,
which start from the poly(A)-tail of the mRNA, to random-primers which are more or less independent
on the quality of the RNA used.

Thus, in principle for whole genome/exon arrays RNA degradation plots are probable useless.
The only reason that 'xps' supports RNA degradation plots for these arrays, too, is that my
implementation is independent of the type of expression array. See also my note in §5.4.1 of
vignette 'xps.pdf'.

Whether RNA degradation plots for whole genome/exon arrays do have any meaning is a question,
which I do not know. Maybe experts can find some tendency when comparing RNA degradation plots
for different whole genome/exon arrays.

Regards,
Christian