Dear Stephen, The normalization process that you outline doesn't make sense, as least to me. In my opinion, you cannot sensibly combine two-dimensional loess (which is what maNorm is doing) with one-dimensional loess (which is what normalizeWithinArrays is doing). You say that this is your first microarray analysis, so I wonder what causes you to want to do it this way. There is no documentation I know of that recommends it. Similarly, no published papers that recommend it. I can't help thinking you're making it unnecessarily hard for yourself. Why not follow the documentation, and do something simple and easy that works? I might also add that two-dimensional loess is a marray package function, not a limma function. And your desired normalization process seems to have nothing to do with modifyWeights. So it's not really my problem! Best wishes Gordon On Thu, 4 Feb 2010, stephen sefick wrote: > There are housekeeping genes that should not be differentially > expressed. I was trying to up weight these in the normalization > process. Also, I am sorry if I am a bother, but what I am trying to > recreate in the normalization process is this: > > background normalization > two dimensional normalization for spatial and then print tip loess > scale normalization between arrays > > #MA <- maNormMain(as(RG, "marrayRaw"), f.loc = list(maNorm2D(weights=w))) > MA <- maNorm(as(RG, "marrayRaw"), norm="twoD") > MA <- normalizeWithinArrays(as(MA, "MAList")) > ################################################################## > > #MA <- normalizeWithinArrays(RG) > > #scale normalization between arrays > WA <- normalizeBetweenArrays(as(MA, "MAList"), method="scale") > > I appreciate everyone's help. I will work on this tomorrow, and try > to form my questions more coherently. > > > thanks, > > Stephen > > On Thu, Feb 4, 2010 at 4:51 PM, Gordon K Smyth <smyth at="" wehi.edu.au=""> wrote: >> >> >>> Date: Wed, 3 Feb 2010 23:31:40 -0600 >>> From: stephen sefick <ssefick at="" gmail.com=""> >>> To: bioc-devel at stat.math.ethz.ch >>> Subject: [Bioc-devel] modifyWeights Problem >>> Content-Type: text/plain; charset=UTF-8 >>> >>> I am using the limma packages for the first time, and I need help with >>> the modifyWeights function. ?I have about three years of experience >>> with R programing, but this is the first time that I have run an >>> analysis with a bioconductor package. ?I have included the code below. >>> I know that this is not a reproducible example and I would gladly >>> give any more information requested. ?I am trying to modify the >>> weights of genes that I know are housekeeping genes (upweight). ?I can >>> not get the modifyWeights function to work to save my life. ?The >>> weights array is 6400, 6. ?The control status vector is 12800- that >>> seems like only two of the arrays are being represented??????? ?I >>> don't know what is wrong. ?Any help would be greatly appreciated >>> thanks, >>> >>> Stephen Sefick >>> >>> library(limma) >>> library(marray) >>> library(convert) >>> library(statmod) >>> #read in targets file >>> targets <- readTargets("targets.txt", row.names="Name") >>> #weight everything with flags !cutoff=0! >>> RG <- read.maimages(targets$FileName, source="genepix", >>> wt.fun=wtflags(cutoff=-50 weight=0)) >>> #read .gal file >>> #remove extra tab in file >>> >>> a <- readGAL() >> >> It is highly unlikely that you need to read a GAL file. ?The limma User's >> Guide tells you that you only need to do this with SPOT input, not with >> GenePix. >> >> It is obvious from the information you give that whatever GAL file you are >> reading, it doesn't match your GenePix files, because it has twice as many >> rows. >> >>> b <- a[,-6] >>> RG$genes<-b >>> #spot types file >>> spottypes <- readSpotTypes() >>> RG$genes$Status <- controlStatus(spottypes, RG$genes) >>> >>> ##################CHANGE weighting for spot types##################### >>> ###################################################################### >>> multiply <- c(1,0,2,0,1,0,1,1,1) >>> ###################################################################### >>> ###################################################################### >>> >>> a <- unique(RG$genes$ID) >>> status=RG$genes$Status >>> w <- modifyWeights(RG$weights, RG$genes$Status, a, c(0, 1)) >> >> Have you looked at the help page for modifyWeights? ?I'd think a quick read >> would tell you that your input arguments aren't the right dimensions, so >> there's no chance of this call working correctly. >> >> I also wonder what you're trying to achieve with modifyWeights, and whether >> you need it at all? ?This function is only needed for special situations. >> >> Best wishes >> Gordon >> >>> -- >>> Stephen Sefick >> >> ______________________________________________________________________ >> The information in this email is confidential and intended solely for the >> addressee. >> You must not disclose, forward, print or use it without the permission of >> the sender. >> ______________________________________________________________________ >> > > > > -- > Stephen Sefick > > Let's not spend our time and resources thinking about things that are > so little or so large that all they really do for us is puff us up and > make us feel like gods. We are mammals, and have not exhausted the > annoying little problems of being mammals. > > -K. Mullis > ______________________________________________________________________ The information in this email is confidential and intend...{{dropped:5}}