Quoting Sean Davis <sdavis2 at="" mail.nih.gov=""> on Thu 27 Sep 2007 00:13:04 BST: > jhs1jjm at leeds.ac.uk wrote: > > Quoting jhs1jjm at leeds.ac.uk on Wed 26 Sep 2007 22:54:01 BST: > > > > > >> Quoting Sean Davis <sdavis2 at="" mail.nih.gov=""> on Wed 26 Sep 2007 17:30:18 BST: > >> > >> > >>> jhs1jjm at leeds.ac.uk wrote: > >>> > >>>> R 2.5.0 on openSUSE 10.2 x86_64. > >>>> Hi, > >>>> > >>>> I'm using the snapCGH package to analyse 2* 244k agilent CGH arrays with > >>>> > >>> the aim > >>> > >>>> of identifying regions of gain/loss. > >>>> So far i've done the following: > >>>> > >>>> > >>>>> targets <- readTargets ("targets.txt") > >>>>> RG1 <-read.maimages (targets$File_names, source="agilent") > >>>>> RG2 <- readPositionalInfo (RG1,source="agilent") > >>>>> RG2$design <- c(-1-1) > >>>>> RG3 <- backgroundCorrect (RG2,method="minimum") > >>>>> MA1 <- normalizeWithinArrays (RG2,method="median") > >>>>> > >>>> then > >>>> > >>>>> MA2 <- > processCGH(MA1,method.of.averaging=mean,ID="MA1$genes$ProbeName") > >>>>> > >>>> Error in order(na.last, decreasing, ...) : > >>>> argument 2 is not a vector > >>>> > >>>> I've looked at ?processCGH and am following the vignette for the snapCGH > >>>> > >>> package > >>> > >>>> fairly closely. Can anyone help with the error. > >>>> > >>> You can't quote variable names like above. I'm not sure that is going > >>> to fix the problem, but until the syntax is correct, it will be hard to > >>> diagnose the issue. > >>> > >>> > >>>> Also i'm unsure of what background correction to use and normalization > >>>> > >>> function > >>> > >>>> (I've been informed that non-linear methods are unsuitable). Also if > >>>> > >> anyone > >> > >>> has > >>> > >>>> any experience of Agilent CGH arrays could they also tell me whether the > >>>> default estimates used for the foreground and background intensities in > >>>> read.maimages are suitable. I'd like to determine the most suitable > >>>> > >> methods > >> > >>>> before as I think the segmentation may take some time on my machine. If > >>>> > >> its > >> > >>> a > >>> > >>>> case of trial and error then then thats fine. Thanks for any input. > >>>> > >>> I would use the LogRatio column of the Agilent file without any further > >>> normalization. The LogRatio is already background corrected. The CGH > >>> algorithms in snapCGH do not depend on the center of the data, so there > >>> isn't really a need to do any further median centering, etc. In fact, > >>> there are probably better methods to center the data, but these use the > >>> segmented data. > >>> > >>> Hope that helps. > >>> > >>> Sean > >>> > >>> > >> Hi Sean, > >> > >> I'm struggling to import the LogRatio column from the Agilent text files. > I'm > >> using read.delim2 but this is bringing my machine to a standstill and > after > >> 45 > >> mins hadn't finished. Is the following the same: > >> > >> > >>> RG1 <- read.maimages(targets$File_names,source="agilent") > >>> RG2 <- readPositionalInfo(RG1,"agilent") > >>> RG2$design <- c(1,-1) > >>> RG3 <- backgroundCorrect(RG2,method="none") > >>> MA1 <- normalizeWithinArrays (RG3,method="none") > >>> LogRatio <- MA1$M > >>> > >> Having just looked at the text file it doesn't appear to be. I've looked > >> through > >> the data import R guide but haven't found anything yet. > >> > >> > > You will probably need to read the read.maimages help pretty carefully. > You will need to specify other columns to read in if you want to read in > the LogRatio column. Alternatively, change the red and green foreground > columns to be rProcessedSignal and gProcessedSignal and then do not do > background correction, as LogRatio is calculated from these. You will > also potentially benefit from looking at the Agilent Feature Extraction > Reference Manual, which explains the columns in the Agilent files. > > http://www.chem.agilent.com/scripts/LiteraturePDF.asp?iWHID=50416 > > > Additionally Sean I tried: > > > > > >> LogRatio <-log2(RG1$R)-log2(RG1$G) > >> > > > > This gives me different results to the text file? > > > > The LogRatio column is calculated from rProcessedSignal and > gProcessedSignal in the Agilent file. These columns are not loaded by > limma by default. > > Hope that helps some. > > Sean Hi Sean, I did the following: #read in the intensity data > RG1 <-read.maimages(targets$File_names,source="agilent", columns=list(R="rProcessedSignal",G="gProcessedSignal")) It sounded like there was an alternative in your email but having looked at the reference manual's column explanation I couldn't see one. #insert info on ch pos of clone into the $genes matrix > RG2 <- readPositionalInfo(RG1,source="agilent") Warning message: NAs introduced by coercion #normalize > MA1 <- normalizeWithinArrays (RG2,method="none") This gives the log2 ratio whereas the agilent text is log10, is this important? Following this i'm getting the same error with processCGH as follows: > MA2 <- processCGH(MA1,ID="ProbeName") Error in order(na.last, decreasing, ...) : argument 2 is not a vector Some of the probes do not have location information, could this be the problem? Thanks again John