Thanks for all your help Jim! On 11/01/11 6:58 AM, "James W. MacDonald" <jmacdon at="" med.umich.edu=""> wrote: > Hi Rick, > > On 1/10/2011 4:57 PM, Rick Frausto wrote: >> Hi Jim, >> >> You're right... >> >>> any(duplicated(unlist(indexProbes(mydata, "both")))) >> [1] TRUE >>> >> >> Figured it would be something simple, almost always is. Guess since the MM >> values are only really necessary for calculating a "real" PM value I should >> generally still be ok with using R Bioconductor packages for downstream >> analysis of these chips?? For example, using eset<-rma() to normalize my >> data should still be ok. > > Yep. RMA only uses PM values, so this will be fine. You only get into > trouble when trying to use mas5 based methods. > >> >> By the way, the documentation on the AffyQCReport function regarding >> signalDist() states that "The first is a boxplot plot of the all pm >> intensities and the second plot consists of kernel density estimates of >> these intensities." From this it would seem to a novice like me that it only >> uses PM values, clearly I'm not correct. I guess these are PM values >> adjusted for the MM signal. > > Nope, they aren't adjusted for MM, they just include the MM values as > well. Here is a little primer on how to see what is going on. > > If you load the affyQCReport package and then type signalDist at the R > prompt, you will get this: > >> signalDist > function (object) > { > par(mfrow = c(2, 1)) > ArrayIndex = as.character(1:length(sampleNames(object))) > boxplot(object, names = ArrayIndex, ylab = "Log2(Intensity)", > xlab = "Array Index") > hist(x = object, lt = 1:length(ArrayIndex), col = 1:length(ArrayIndex), > which = "both") > temppar <- par() > legend(((temppar$xaxp[2] - temppar$xaxp[1])/temppar$xaxp[3]) * > (temppar$xaxp[3] - 1) + temppar$xaxp[1], temppar$yaxp[2], > as.character(ArrayIndex), lt = 1:length(ArrayIndex), > col = 1:length(ArrayIndex), cex = 0.5) > } > <environment: namespace:affyqcreport=""> > > So you can see that we are calling boxplot() as well as hist() on the > 'object', which is an AffyBatch. Let's see what boxplot() and hist() do. > >> boxplot > standardGeneric for "boxplot" defined from package "graphics" > > function (x, ...) > standardGeneric("boxplot") > <environment: 0x184ea378=""> > Methods may be defined for arguments: x > Use showMethods("boxplot") for currently available ones. > > So this is an S4 method, and the methods are slightly harder to get to, > but let's follow the prescription on the last line. > >> showMethods(boxplot, class = "AffyBatch", includeDefs = TRUE) > Function: boxplot (package graphics) > x="AffyBatch" > function (x, ...) > { > .local <- function (x, which = "both", range = 0, main, ...) > { > tmp <- description(x) > if (missing(main) && (is(tmp, "MIAME"))) > main <- tmp at title > tmp <- unlist(indexProbes(x, which)) > tmp <- tmp[seq(1, length(tmp), len = 5000)] > boxplot(data.frame(log2(intensity(x)[tmp, ])), main = main, > range = range, ...) > } > .local(x, ...) > } > > Note two things here. I added in class = "AffyBatch", because there may > be other boxplot methods for other objects, and we really don't care > about them. Additionally, I included includeDefs = TRUE, which will > cause the function to be output. > > The .local function has a default of which = 'both', and you see that > argument is used for the call to indexProbes (also note that there is a > '...' argument to .local, that could be used to pass in a which = "pm" > in signalDist() to override the default, but it is not, so the help page > is incorrect). If you look at ?indexProbes, you will see this in the > methods section: > > indexProbes 'signature(object = "AffyBatch", which = > "character")': returns a list with locations of the probes in > each probe set. The affyID corresponding to the probe set to > retrieve can be specified in an optional parameter > 'genenames'. By default, all the affyIDs are retrieved. The > names of the elements in the list returned are the affyIDs. > 'which' can be "pm", "mm", or "both". If "both" then perfect > match locations are given followed by mismatch locations. > > The warning you get comes from here: > > tmp <- unlist(indexProbes(x, which)) > tmp <- tmp[seq(1, length(tmp), len = 5000)] > boxplot(data.frame(log2(intensity(x)[tmp, ])), main = main, > range = range, ...) > > Which is basically getting a subset of 5000 probes to create the > boxplot. Since half of your indices from indexProbes() will be NA, a > bunch of the tmp variable will be NAs as well. We can re-create the > warning you get below with a little example: > >> x <- matrix(rnorm(100), ncol = 10) >> row.names(x) <- letters[1:10] >> z <- data.frame(x[c(1,2,3,NA,4,5,NA),]) > Warning message: > In data.row.names(row.names, rowsi, i) : > some row.names duplicated: 7 --> row.names NOT used > > Best, > > Jim > > >> >> Thanks for figuring this out for me. Let me know if these and other related >> questions would be better served as standalone e-mails. >> >> Cheers, >> Rick >> >> >> >> On 10/01/11 7:04 AM, "James W. MacDonald"<jmacdon at="" med.umich.edu=""> wrote: >> >>> Hi Rick, >>> >>> After all that, the reason is really simple. You are trying to use >>> affyQCReport on a PM-only chip, which isn't going to work out so well. I >>> don't have any mogene data around to play with (and don't have the time >>> to go searching), so I will have to make some educated guesses. >>> >>> Internally in signalDist() you are calling boxplot() and hist() on your >>> AffyBatch. And the default for both functions is to use both PM and MM >>> probes. I'm betting that >>> >>> any(duplicated(unlist(indexProbes(mydata, "both")))) >>> >>> returns TRUE, indicating that indexProbes doesn't work correctly on a >>> PM-only chip, which is fair enough, as it was never designed to do so. >>> >>> And plot(qc(mydata)) will never work, as it relies on computing a >>> Wilcoxon signed-rank between the PM and MM probes, and since you don't >>> have MM probes, well you get the picture... >>> >>> Best, >>> >>> Jim >>> >>> >>> >>> On 1/7/2011 6:56 PM, Rick Frausto wrote: >>>> Hi Jim, >>>> >>>> Ok, so after doing a bit of reading and re-reading I was eventually able to >>>> generate each page in a quartz window that the "QCReport" function should >>>> also generate. I found which ones give me the errors. So, there should be 6 >>>> pages in total. Page 2 gives me the duplication error and page 3 gives me >>>> the error in evaluating the argument x. The other pages are ok and are >>>> generated as expected. >>>> >>>> In brief, page 2 is suppose to be generated with the "signalDist(mydata)" >>>> command. Page 3 is suppose to generated with the "plot(qc(mydata))" >>>> command. >>>> >>>> So, I guess there must be particular requirements for these commands that >>>> I'm missing.I've included the session below along with traceback() and >>>> sessionInfo(). >>>> >>>> >>>> R version 2.12.0 (2010-10-15) >>>> Copyright (C) 2010 The R Foundation for Statistical Computing >>>> ISBN 3-900051-07-0 >>>> Platform: x86_64-apple-darwin9.8.0/x86_64 (64-bit) >>>> >>>> R is free software and comes with ABSOLUTELY NO WARRANTY. >>>> You are welcome to redistribute it under certain conditions. >>>> Type 'license()' or 'licence()' for distribution details. >>>> >>>> Natural language support but running in an English locale >>>> >>>> R is a collaborative project with many contributors. >>>> Type 'contributors()' for more information and >>>> 'citation()' on how to cite R or R packages in publications. >>>> >>>> Type 'demo()' for some demos, 'help()' for on-line help, or >>>> 'help.start()' for an HTML browser interface to help. >>>> Type 'q()' to quit R. >>>> >>>> [R.app GUI 1.35 (5632) x86_64-apple-darwin9.8.0] >>>> >>>> [Workspace restored from /Users/rickfrausto/.RData] >>>> [History restored from /Users/rickfrausto/.Rapp.history] >>>> >>>>> library(simpleaffy) >>>> Loading required package: affy >>>> Loading required package: Biobase >>>> >>>> Welcome to Bioconductor >>>> >>>> Vignettes contain introductory material. To view, type >>>> 'openVignette()'. To cite Bioconductor, see >>>> 'citation("Biobase")' and for packages 'citation(pkgname)'. >>>> >>>> Loading required package: genefilter >>>> Loading required package: gcrma >>>> >>>> Attaching package: 'simpleaffy' >>>> >>>> The following object(s) are masked _by_ '.GlobalEnv': >>>> >>>> getBioC >>>> >>>>> library(affy) >>>>> mydata<- ReadAffy() >>>>> eset<- rma(mydata) >>>> Background correcting >>>> Normalizing >>>> Calculating Expression >>>>> library(affycoretools); affystart(plot=T, express="rma") >>>> Loading required package: GO.db >>>> Loading required package: AnnotationDbi >>>> Loading required package: DBI >>>> Loading required package: KEGG.db >>>> Background correcting >>>> Normalizing >>>> Calculating Expression >>>> Please give the x-coordinate for a legend.30 >>>> Please give the y-coordinate for a legend.80 >>>> ExpressionSet (storageMode: lockedEnvironment) >>>> assayData: 34760 features, 35 samples >>>> element names: exprs >>>> protocolData >>>> sampleNames: A_WT1_NT_2hr.CEL B_WT1_NT_2hr.CEL ... >>>> ZI_ST1KO_HIL6_12hr.CEL (35 total) >>>> varLabels: ScanDate >>>> varMetadata: labelDescription >>>> phenoData >>>> sampleNames: A_WT1_NT_2hr.CEL B_WT1_NT_2hr.CEL ... >>>> ZI_ST1KO_HIL6_12hr.CEL (35 total) >>>> varLabels: sample >>>> varMetadata: labelDescription >>>> featureData: none >>>> experimentData: use 'experimentData(object)' >>>> Annotation: mogene10stv1 >>>>> write.exprs(eset, file="mydata.txt") >>>>> x<- data.frame(exprs(eset), exprs(eset_PMA), assayDataElement(eset_PMA, >>>> "se.exprs")); x<- x[,sort(names(x))]; write.table(x, file="mydata_PMA.xls", >>>> quote=F, col.names = NA, sep="\t") >>>> Error in exprs(eset_PMA) : >>>> error in evaluating the argument 'object' in selecting a method for >>>> function 'exprs' >>>>> mypm<- pm(mydata) >>>>> mymm<- mm(mydata) >>>>> myaffyids<- probeNames(mydata) >>>>> result<- data.frame(myaffyids, mypm, mymm) >>>>> eset; pData(eset) >>>> ExpressionSet (storageMode: lockedEnvironment) >>>> assayData: 34760 features, 35 samples >>>> element names: exprs >>>> protocolData >>>> sampleNames: A_WT1_NT_2hr.CEL B_WT1_NT_2hr.CEL ... >>>> ZI_ST1KO_HIL6_12hr.CEL (35 total) >>>> varLabels: ScanDate >>>> varMetadata: labelDescription >>>> phenoData >>>> sampleNames: A_WT1_NT_2hr.CEL B_WT1_NT_2hr.CEL ... >>>> ZI_ST1KO_HIL6_12hr.CEL (35 total) >>>> varLabels: sample >>>> varMetadata: labelDescription >>>> featureData: none >>>> experimentData: use 'experimentData(object)' >>>> Annotation: mogene10stv1 >>>> sample >>>> A_WT1_NT_2hr.CEL 1 >>>> B_WT1_NT_2hr.CEL 2 >>>> C_WT1_NT_12hr.CEL 3 >>>> D_WT1_NT_12hr.CEL 4 >>>> E_WT1_HIL6_2hr.CEL 5 >>>> F_WT1_HIL6_2hr.CEL 6 >>>> G_WT1_HIL6_12hr.CEL 7 >>>> H_WT1_HIL6_12hr.CEL 8 >>>> I_FF_NT_2hr.CEL 9 >>>> J_FF_NT_2hr.CEL 10 >>>> K_FF_NT_12hr.CEL 11 >>>> L_FF_NT_12hr.CEL 12 >>>> M_FF_HIL6_2hr.CEL 13 >>>> N_FF_HIL6_2hr.CEL 14 >>>> O_FF_HIL6_12hr.CEL 15 >>>> P_FF_HIL6_12hr.CEL 16 >>>> Q_WT2_NT_2hr.CEL 17 >>>> R_WT2_NT_2hr.CEL 18 >>>> S_WT2_NT_12hr.CEL 19 >>>> T_WT2_NT_12hr.CEL 20 >>>> U_WT2_HIL6_2hr.CEL 21 >>>> V_WT2_HIL6_2hr.CEL 22 >>>> W_WT2_HIL6_12hr.CEL 23 >>>> X_WT2_HIL6_12hr.CEL 24 >>>> Y_DD_NT_2hr.CEL 25 >>>> Z_DD_NT_2hr.CEL 26 >>>> ZA_DD_NT_12hr.CEL 27 >>>> ZB_DD_NT_12hr.CEL 28 >>>> ZC_DD_HIL6_2hr.CEL 29 >>>> ZD_DD_HIL6_2hr.CEL 30 >>>> ZE_DD_HIL6_12hr.CEL 31 >>>> ZF_DD_HIL6_12hr.CEL 32 >>>> ZG_ST1KO_NT_2hr.CEL 33 >>>> ZH_ST1KO_HIL6_2hr.CEL 34 >>>> ZI_ST1KO_HIL6_12hr.CEL 35 >>>>> data.frame(eset) >>>> X10338001 X10338003 X10338004 X10338017 X10338025 >>>> A_WT1_NT_2hr.CEL 11.71717 10.183620 9.440631 12.79412 8.823529 >>>> B_WT1_NT_2hr.CEL 11.78778 10.027760 9.489226 12.98544 8.843002 >>>> X10338026 X10338029 X10338035 X10338036 X10338037 >>>> A_WT1_NT_2hr.CEL 13.22585 9.405038 8.853564 9.379031 3.661987 >>>> B_WT1_NT_2hr.CEL 13.29043 9.575309 8.772872 9.513050 3.514885 >>>> X10338041 X10338042 X10338044 X10338047 X10338056 >>>> A_WT1_NT_2hr.CEL 10.94638 10.116516 11.88296 8.872839 3.133222 >>>> B_WT1_NT_2hr.CEL 11.23276 10.134084 12.03381 7.568584 3.088548 >>>> X10338059 X10338060 X10338063 X10338064 X10338065 >>>> >>>> JIM, I TRUNCATED THIS LIST, BUT THOUGHT IT MIGHT BE USEFUL IN DIAGNOSING >>>> THE >>>> PROBLEMS I'M HAVING. SESSION IS CONTINUED BELOW. >>>> >>>>> library(affyQCReport) >>>> Loading required package: lattice >>>>> titlePage(mydata) >>>> [1] TRUE >>>>> signalDist(mydata) >>>> Warning message: >>>> In data.row.names(row.names, rowsi, i) : >>>> some row.names duplicated: >>>> 4,8,9,13,14,15,16,24,25,26,27,28,29,30,31,36,37,38,39,47,48,49,50,51,5 2,53,>>>> 5 >>>> 4,58,59,60,64,65,66,83,84,85,86,87,88,89,90,91,92,93,94,95,96,97,98,99 ,102,>>>> 1 >>>> 03,104,108,109,110,111,114,119,120,121,122,127,134,136,137,138,139,141 ,142,>>>> 1 >>>> 47,148,149,152,153,156,157,158,159,162,163,164,165,166,167,168,169,170 ,171,>>>> 1 >>>> 73,175,176,179,180,183,184,185,186,191,192,195,197,198,199,200,202,206 ,207,>>>> 2 >>>> 10,219,220,227,228,229,230,233,234,235,240,241,243,245,246,248,249,250 ,251,>>>> 2 >>>> 52,253,257,259,260,266,271,272,276,277,280,281,284,286,287,289,290,291 ,292,>>>> 2 >>>> 96,297,298,302,304,305,306,310,311,312,313,317,318,319,321,322,324,334 ,337,>>>> 3 >>>> 38,339,340,341,345,346,350,351,356,359,362,364,366,367,370,371,373,376 ,378,>>>> 3 >>>> 82,383,384,385,386,387,388,389,391,394,395,397,398,399,400,402,403,405 ,406,>>>> 4 >>>> 07,409,410,411,415,416,418,419,425,431,432,433,434,435,440,441,443,445 ,447,>>>> 4 >>>> 49,450,452,454,455,456,461,464,466,470,472,473,481,487,488,491,492,493 ,494,>>>> 4 >>>> 95,496,497,498,499,501,502,504,506,507,509,511,513,515,516,51 [... >>>> truncated] >>>>> plot(qc(mydata)) >>>> Error in plot(qc(mydata)) : >>>> error in evaluating the argument 'x' in selecting a method for function >>>> 'plot' >>>>> borderQC1(mydata) >>>> [1] TRUE >>>>> borderQC2(mydata) >>>> [1] TRUE >>>>> correlationPlot(mydata) >>>> [1] TRUE >>>>> titlePage(mydata) >>>> [1] TRUE >>>>> titlePage(mydata) >>>> Error in polygon(c(0, 0, 0.9, 0.9, 0), c(0.05, 0.95, 0.95, 0.05, 0.05)) : >>>> plot.new has not been called yet >>>>> correlationPlot(mydata) >>>> [1] TRUE >>>>> titlePage(mydata) >>>> Error in polygon(c(0, 0, 0.9, 0.9, 0), c(0.05, 0.95, 0.95, 0.05, 0.05)) : >>>> plot.new has not been called yet >>>> In addition: Warning message: >>>> Display list redraw incomplete >>>>> borderQC1(mydata) >>>> [1] TRUE >>>>> titlePage(mydata) >>>> [1] TRUE >>>>> titlePage(mydata) >>>> Error in polygon(c(0, 0, 0.9, 0.9, 0), c(0.05, 0.95, 0.95, 0.05, 0.05)) : >>>> plot.new has not been called yet >>>>> traceback() >>>> 2: polygon(c(0, 0, 0.9, 0.9, 0), c(0.05, 0.95, 0.95, 0.05, 0.05)) >>>> 1: titlePage(mydata) >>>>> sessionInfo() >>>> R version 2.12.0 (2010-10-15) >>>> Platform: x86_64-apple-darwin9.8.0/x86_64 (64-bit) >>>> >>>> locale: >>>> [1] en_AU.UTF-8/en_AU.UTF-8/C/C/en_AU.UTF-8/en_AU.UTF-8 >>>> >>>> attached base packages: >>>> [1] stats graphics grDevices utils datasets methods base >>>> >>>> other attached packages: >>>> [1] affyQCReport_1.28.1 lattice_0.19-13 affycoretools_1.22.0 >>>> [4] KEGG.db_2.4.5 GO.db_2.4.5 RSQLite_0.9-4 >>>> [7] DBI_0.2-5 AnnotationDbi_1.12.0 mogene10stv1cdf_2.7.0 >>>> [10] simpleaffy_2.26.1 gcrma_2.22.0 genefilter_1.32.0 >>>> [13] affy_1.28.0 Biobase_2.10.0 >>>> >>>> loaded via a namespace (and not attached): >>>> [1] affyio_1.18.0 affyPLM_1.26.0 annaffy_1.22.0 >>>> [4] annotate_1.28.0 biomaRt_2.6.0 Biostrings_2.18.2 >>>> [7] Category_2.16.0 GOstats_2.16.0 graph_1.28.0 >>>> [10] grid_2.12.0 GSEABase_1.12.2 IRanges_1.8.7 >>>> [13] limma_3.6.9 preprocessCore_1.12.0 RBGL_1.26.0 >>>> [16] RColorBrewer_1.0-2 RCurl_1.4-3 splines_2.12.0 >>>> [19] survival_2.36-2 tools_2.12.0 XML_3.2-0 >>>> [22] xtable_1.5-6 >>>>> >>>> >>>> On 7/01/11 12:47 PM, "James W. MacDonald"<jmacdon at="" med.umich.edu=""> wrote: >>>> >>>>> Hi Rick, >>>>> >>>>> What happens if you load the simpleaffy package first? >>>>> >>>>> Best, >>>>> >>>>> Jim >>>>> >>>>> On 1/7/2011 2:14 PM, Rick Frausto wrote: >>>>>> Hi James, >>>>>> >>>>>> Below is the information that you requested - traceback() and >>>>>> sessioninfo(). >>>>>> Doesn't seem like much to me, but perhaps you can help. As you answer to >>>>>> a >>>>>> lot of e-mails, thought I'd remind you that this is in regards to the >>>>>> "some >>>>>> row.names duplicated" error. >>>>>> >>>>>> Hope your holidays were good! >>>>>> >>>>>> -Rick >>>>>> >>>>>> [R.app GUI 1.35 (5632) x86_64-apple-darwin9.8.0] >>>>>> >>>>>> [Workspace restored from /Users/rickfrausto/.RData] >>>>>> [History restored from /Users/rickfrausto/.Rapp.history] >>>>>> >>>>>>> library(affy) >>>>>> Loading required package: Biobase >>>>>> >>>>>> Welcome to Bioconductor >>>>>> >>>>>> Vignettes contain introductory material. To view, type >>>>>> 'openVignette()'. To cite Bioconductor, see >>>>>> 'citation("Biobase")' and for packages 'citation(pkgname)'. >>>>>> >>>>>>> mydata<- ReadAffy() >>>>>>> eset<- rma(mydata) >>>>>> Background correcting >>>>>> Normalizing >>>>>> Calculating Expression >>>>>>> write.exprs(eset, file="mydata.txt") >>>>>>> mypm<- pm(mydata) >>>>>>> mymm<- mm(mydata) >>>>>>> myaffyids<- probeNames(mydata) >>>>>>> result<- data.frame(myaffyids, mypm, mymm) >>>>>>> library(affyQCReport); QCReport(mydata, file="ExampleQC.pdf") >>>>>> Loading required package: lattice >>>>>> Warning message: >>>>>> In data.row.names(row.names, rowsi, i) : >>>>>> some row.names duplicated: >>>>>> >> 4,8,9,13,14,15,16,24,25,26,27,28,29,30,31,36,37,38,39,47,48,49,50,5 1,52,53,>> >> >> >> 5 >>>>>> >> 4,58,59,60,64,65,66,83,84,85,86,87,88,89,90,91,92,93,94,95,96,97,98 ,99,102,>> >> >> >> 1 >>>>>> >> 03,104,108,109,110,111,114,119,120,121,122,127,134,136,137,138,139, 141,142,>> >> >> >> 1 >>>>>> >> 47,148,149,152,153,156,157,158,159,162,163,164,165,166,167,168,169, 170,171,>> >> >> >> 1 >>>>>> >> 73,175,176,179,180,183,184,185,186,191,192,195,197,198,199,200,202, 206,207,>> >> >> >> 2 >>>>>> >> 10,219,220,227,228,229,230,233,234,235,240,241,243,245,246,248,249, 250,251,>> >> >> >> 2 >>>>>> >> 52,253,257,259,260,266,271,272,276,277,280,281,284,286,287,289,290, 291,292,>> >> >> >> 2 >>>>>> >> 96,297,298,302,304,305,306,310,311,312,313,317,318,319,321,322,324, 334,337,>> >> >> >> 3 >>>>>> >> 38,339,340,341,345,346,350,351,356,359,362,364,366,367,370,371,373, 376,378,>> >> >> >> 3 >>>>>> >> 82,383,384,385,386,387,388,389,391,394,395,397,398,399,400,402,403, 405,406,>> >> >> >> 4 >>>>>> >> 07,409,410,411,415,416,418,419,425,431,432,433,434,435,440,441,443, 445,447,>> >> >> >> 4 >>>>>> >> 49,450,452,454,455,456,461,464,466,470,472,473,481,487,488,491,492, 493,494,>> >> >> >> 4 >>>>>> 95,496,497,498,499,501,502,504,506,507,509,511,513,515,516,51 [... >>>>>> truncated] >>>>>> Error in plot(qc(object)) : >>>>>> error in evaluating the argument 'x' in selecting a method for >>>>>> function >>>>>> 'plot' >>>>>>> traceback() >>>>>> 2: plot(qc(object)) >>>>>> 1: QCReport(mydata, file = "ExampleQC.pdf") >>>>>>> sessionInfo() >>>>>> R version 2.12.0 (2010-10-15) >>>>>> Platform: x86_64-apple-darwin9.8.0/x86_64 (64-bit) >>>>>> >>>>>> locale: >>>>>> [1] en_AU.UTF-8/en_AU.UTF-8/C/C/en_AU.UTF-8/en_AU.UTF-8 >>>>>> >>>>>> attached base packages: >>>>>> [1] stats graphics grDevices utils datasets methods base >>>>>> >>>>>> other attached packages: >>>>>> [1] affyQCReport_1.28.1 latptice_0.19-13 mogene10stv1cdf_2.7.0 >>>>>> [4] affy_1.28.0 Biobase_2.10.0 >>>>>> >>>>>> loaded via a namespace (and not attached): >>>>>> [1] affyio_1.18.0 affyPLM_1.26.0 annotate_1.28.0 >>>>>> [4] AnnotationDbi_1.12.0 Biostrings_2.18.2 DBI_0.2-5 >>>>>> [7] gcrma_2.22.0 genefilter_1.32.0 grid_2.12.0 >>>>>> [10] IRanges_1.8.7 preprocessCore_1.12.0 RColorBrewer_1.0-2 >>>>>> [13] RSQLite_0.9-4 simpleaffy_2.26.1 splines_2.12.0 >>>>>> [16] survival_2.36-2 tools_2.12.0 xtable_1.5-6 >>>>>>> >>>>>> >>>>>> >>>>>> >>>>>> >>>>>> On 20/12/10 6:33 AM, "James W. MacDonald"<jmacdon at="" med.umich.edu=""> >>>>>> wrote: >>>>>> >>>>>>> Hi Rick, >>>>>>> >>>>>>> On 12/17/2010 9:24 PM, Rick Frausto wrote: >>>>>>>> Hey Jim, >>>>>>>> >>>>>>>> Ok, I will give that a go. The only problem is an ExpressionSet >>>>>>>> contains >>>>>>>> all >>>>>>>> of the necessary information for further analysis (e.g. phenodata, >>>>>>>> featuredata and annotation, etc - including, treatment type, cell type, >>>>>>>> time >>>>>>>> points, replicates). I am still learning how to include all of these >>>>>>>> for >>>>>>>> a >>>>>>>> complete ExpressionSet. As a starting point I've loaded a txt file >>>>>>>> containing some of this information (gene abbrev, ontology, probeset >>>>>>>> ID) >>>>>>>> which I created using Affymetrix's Expression Console software, without >>>>>>>> replicate, time point and cell type info. Doing this I've gotten as far >>>>>>>> as >>>>>>>> creating a minimal ExpressionSet, which I guess the functions you >>>>>>>> mention >>>>>>>> below do just that but with the information contained in the CEL file >>>>>>>> only. >>>>>>>> >>>>>>>> In any case, since as you say, the functions in the online manual >>>>>>>> create >>>>>>>> a >>>>>>>> proper ExpressionSet why would I get the issue of duplication? >>>>>>> >>>>>>> Oh yeah, the original question ;-D. Try running QCreport() again, and >>>>>>> when it errors out run traceback() and send the output. Also include the >>>>>>> output of sessionInfo(). >>>>>>> >>>>>>> Jim >>>>>>> >>>>>>> >>>>>>>> >>>>>>>> In regards to the 64-bit discussion. It may have very well made enough >>>>>>>> of >>>>>>>> a >>>>>>>> difference as it did not come up with the memory error the last time I >>>>>>>> tried >>>>>>>> it. Going to upgrade to 8GB RAM anyways, can't hurt. >>>>>>>> >>>>>>>> Cheers, >>>>>>>> Rick >>>>>>>> >>>>>>>> >>>>>>>> On 17/12/10 7:20 AM, "James W. MacDonald"<jmacdon at="" med.umich.edu=""> >>>>>>>> wrote: >>>>>>>> >>>>>>>>> Hi Rick, >>>>>>>>> >>>>>>>>> On 12/16/2010 4:13 PM, Rick Frausto wrote: >>>>>>>>>> Hi Jim, >>>>>>>>>> >>>>>>>>>> How do I run an RMA analysis without a proper ExpresionSet? Honest >>>>>>>>>> answer, >>>>>>>>>> I >>>>>>>>>> don't know, I just put in a command line from a manual I found online >>>>>>>>>> and >>>>>>>>>> it >>>>>>>>>> spit out some result- see #3 Affy packages in following link ( >>>>>>>>>> http://manuals.bioinformatics.ucr.edu/home/R_BioCondManual#biocon_int >>>>>>>>>> ro >>>>>>>>>> ). >>>>>>>>> >>>>>>>>> You are mistaken. All of the functions mentioned there result in a >>>>>>>>> proper ExpressionSet. And if you just do >>>>>>>>> >>>>>>>>> abatch<- ReadAffy() >>>>>>>>> eset<- rma(abatch) >>>>>>>>> >>>>>>>>> Then you will 100% surely get an ExpressionSet. >>>>>>>>> >>>>>>>>>> >>>>>>>>>> Perhaps you don't need an ExpressionSet until after the >>>>>>>>>> preprocessing, >>>>>>>>>> at >>>>>>>>>> least that is what I get from the "An Introduction to Bioconductor's >>>>>>>>>> ExpressionSet Class" written by Seth Falcon, Martin Morgan and Robert >>>>>>>>>> Gentleman. Everything seemed to be going smoothly until I tried to >>>>>>>>>> get >>>>>>>>>> a >>>>>>>>>> QC >>>>>>>>>> Report. >>>>>>>>>> >>>>>>>>>> Now, the answer for why I would want to do such a thing is easy. >>>>>>>>>> Simply >>>>>>>>>> that >>>>>>>>>> I don't know any better :) Just started working with R a few days >>>>>>>>>> ago, >>>>>>>>>> but >>>>>>>>>> I'm learning. >>>>>>>>>> >>>>>>>>>> >>>>>>>>>> Apparently Snow Leopard running on 32bit can only utilize about 3.2GB >>>>>>>>>> of >>>>>>>>>> RAM, whereas 64bit can make use of all 4GB. I'll switch to the 64 bit >>>>>>>>>> OS >>>>>>>>>> and >>>>>>>>>> see if it makes a difference. >>>>>>>>> >>>>>>>>> Well, it won't be much different. The reason a 32-bit OS can only use >>>>>>>>> about 3.2 Gb of RAM is that the OS needs some to run. The 64-bit OS >>>>>>>>> also >>>>>>>>> needs to use some RAM, so you won't get all 4 Gb there either. The >>>>>>>>> issue >>>>>>>>> is how much RAM can be allocated to a single process, and on a 64-bit >>>>>>>>> OS >>>>>>>>> that gets bumped up significantly. >>>>>>>>> >>>>>>>>> Best, >>>>>>>>> >>>>>>>>> Jim >>>>>>>>> >>>>>>>>> >>>>>>>>> >>>>>>>>>> >>>>>>>>>> Thanks for your insight! >>>>>>>>>> >>>>>>>>>> Cheers, >>>>>>>>>> Rick >>>>>>>>>> >>>>>>>>>> >>>>>>>>>> >>>>>>>>>> >>>>>>>>>> On 16/12/10 11:31 AM, "James W. MacDonald"<jmacdon at="" med.umich.edu=""> >>>>>>>>>> wrote: >>>>>>>>>> >>>>>>>>>>> Hi Rick, >>>>>>>>>>> >>>>>>>>>>> On 12/16/2010 12:57 PM, Rick Frausto wrote: >>>>>>>>>>>> Thanks Jim! How much memory would I need, I currently have 4GB, but >>>>>>>>>>>> have >>>>>>>>>>>> quite a few other programs running in the background...I'll see if >>>>>>>>>>>> closing >>>>>>>>>>>> them helps. Perhaps setting up an "ExpressionSet" would solve the >>>>>>>>>>>> problem. >>>>>>>>>>>> I >>>>>>>>>>>> just started reading up on how to set one of these up yesterday. >>>>>>>>>>>> Will >>>>>>>>>>>> do >>>>>>>>>>>> this and see if the duplicates will go away. >>>>>>>>>>>> >>>>>>>>>>>> The "mydata" originates from CEL files and then I run the RMA >>>>>>>>>>>> analysis >>>>>>>>>>>> on >>>>>>>>>>>> it, but I didn't actually set up a proper ExpressionSet. I'm >>>>>>>>>>>> guessing >>>>>>>>>>>> that >>>>>>>>>>>> doing this might reduce the QCReport PDF file size quite >>>>>>>>>>>> considerably >>>>>>>>>>>> since >>>>>>>>>>>> I won't have any duplication and will make further analysis easier. >>>>>>>>>>> >>>>>>>>>>> How do you run an RMA analysis without setting up a proper >>>>>>>>>>> ExpressionSet? The default behavior is to create one. In addition, >>>>>>>>>>> why >>>>>>>>>>> would you want to do such a thing? The ExpressionSet class is >>>>>>>>>>> specifically designed to contain these sorts of data. >>>>>>>>>>> >>>>>>>>>>> >>>>>>>>>>>> >>>>>>>>>>>> I'm running Snow Leopard OSX which can be set up as 64bit. Would >>>>>>>>>>>> running >>>>>>>>>>>> as >>>>>>>>>>>> 64bit still necessitate more RAM? >>>>>>>>>>> >>>>>>>>>>> Probably. The difference isn't efficiency, but the ability to >>>>>>>>>>> address >>>>>>>>>>> more RAM. A 32-bit OS can still address all the available memory >>>>>>>>>>> that >>>>>>>>>>> you will have with just 4 Gb RAM, so you need to bump that up if you >>>>>>>>>>> want to do all the chips together. As for how much, I don't know. >>>>>>>>>>> Since >>>>>>>>>>> RAM isn't that expensive these days, you might look at maxing your >>>>>>>>>>> box >>>>>>>>>>> out. >>>>>>>>>>> >>>>>>>>>>> Best, >>>>>>>>>>> >>>>>>>>>>> Jim >>>>>>>>>>> >>>>>>>>>>> >>>>>>>>>>> >>>>>>>>>>> >>>>>>>>>>>> >>>>>>>>>>>> Thanks again, >>>>>>>>>>>> Rick >>>>>>>>>>>> >>>>>>>>>>>> >>>>>>>>>>>> On 15/12/10 7:45 AM, "James W. MacDonald"<jmacdon at="" med.umich.edu=""> >>>>>>>>>>>> wrote: >>>>>>>>>>>> >>>>>>>>>>>> Hi Rick, >>>>>>>>>>>> >>>>>>>>>>>> On 12/14/2010 3:55 PM, Rick Frausto wrote: >>>>>>>>>>>> Dear All, >>>>>>>>>>>> >>>>>>>>>>>> I have recently entered the world of R. Through some trial and >>>>>>>>>>>> error >>>>>>>>>>>> I'm >>>>>>>>>>>> becoming more familiar with R and the relevant Bioconductor Affy >>>>>>>>>>>> packages. >>>>>>>>>>>> I?m a molecular and cell biologist with rudimentary statistical >>>>>>>>>>>> knowledge >>>>>>>>>>>> and even less knowledge with respect to R. >>>>>>>>>>>> >>>>>>>>>>>> When I enter the following: >>>>>>>>>>>> >>>>>>>>>>>> library(affyQCReport); QCReport(mydata, file="ExampleQC.pdf") >>>>>>>>>>>> >>>>>>>>>>>> I get some errors in return. >>>>>>>>>>>> >>>>>>>>>>>> Loading required package: lattice >>>>>>>>>>>> Error: cannot allocate vector of size 437.4 Mb >>>>>>>>>>>> >>>>>>>>>>>> This indicates that you need more RAM, as you are running out of >>>>>>>>>>>> memory. >>>>>>>>>>>> >>>>>>>>>>>> In addition: Warning message: >>>>>>>>>>>> In data.row.names(row.names, rowsi, i) : >>>>>>>>>>>> some row.names duplicated: >>>>>>>>>>>> >>>>>>>>>> 4,8,9,13,14,15,16,24,25,26,27,28,29,30,31,36,37,38,39,47,48,49,50,51, >>>>>>>>>> 52 >>>>>>>>>> ,5 >>>>>>>>>> 3, >>>>>>>>>>>> >>>>>>>>>>>> >>>>>>>>>> 5 >>>>>>>>>>>> >>>>>>>>>> 4,58,59,60,64,65,66,83,84,85,86,87,88,89,90,91,92,93,94,95,96,97,98,9 >>>>>>>>>> 9, >>>>>>>>>> 10 >>>>>>>>>> 2, >>>>>>>>>>>> >>>>>>>>>>>> >>>>>>>>>> 1 >>>>>>>>>>>> >>>>>>>>>> 03,104,108,109,110,111,114,119,120,121,122,127,134,136,137,138,139,14 >>>>>>>>>> 1, >>>>>>>>>> 14 >>>>>>>>>> 2, >>>>>>>>>>>> >>>>>>>>>>>> >>>>>>>>>> 1 >>>>>>>>>>>> >>>>>>>>>> 47,148,149,152,153,156,157,158,159,162,163,164,165,166,167,168,169,17 >>>>>>>>>> 0, >>>>>>>>>> 17 >>>>>>>>>> 1, >>>>>>>>>>>> >>>>>>>>>>>> >>>>>>>>>> 1 >>>>>>>>>>>> >>>>>>>>>> 73,175,176,179,180,183,184,185,186,191,192,195,197,198,199,200,202,20 >>>>>>>>>> 6, >>>>>>>>>> 20 >>>>>>>>>> 7, >>>>>>>>>>>> >>>>>>>>>>>> >>>>>>>>>> 2 >>>>>>>>>>>> >>>>>>>>>> 10,219,220,227,228,229,230,233,234,235,240,241,243,245,246,248,249,25 >>>>>>>>>> 0, >>>>>>>>>> 25 >>>>>>>>>> 1, >>>>>>>>>>>> >>>>>>>>>>>> >>>>>>>>>> 2 >>>>>>>>>>>> >>>>>>>>>> 52,253,257,259,260,266,271,272,276,277,280,281,284,286,287,289,290,29 >>>>>>>>>> 1, >>>>>>>>>> 29 >>>>>>>>>> 2, >>>>>>>>>>>> >>>>>>>>>>>> >>>>>>>>>> 2 >>>>>>>>>>>> >>>>>>>>>> 96,297,298,302,304,305,306,310,311,312,313,317,318,319,321,322,324,33 >>>>>>>>>> 4, >>>>>>>>>> 33 >>>>>>>>>> 7, >>>>>>>>>>>> >>>>>>>>>>>> >>>>>>>>>> 3 >>>>>>>>>>>> >>>>>>>>>> 38,339,340,341,345,346,350,351,356,359,362,364,366,367,370,371,373,37 >>>>>>>>>> 6, >>>>>>>>>> 37 >>>>>>>>>> 8, >>>>>>>>>>>> >>>>>>>>>>>> >>>>>>>>>> 3 >>>>>>>>>>>> >>>>>>>>>> 82,383,384,385,386,387,388,389,391,394,395,397,398,399,400,402,403,40 >>>>>>>>>> 5, >>>>>>>>>> 40 >>>>>>>>>> 6, >>>>>>>>>>>> >>>>>>>>>>>> >>>>>>>>>> 4 >>>>>>>>>>>> >>>>>>>>>> 07,409,410,411,415,416,418,419,425,431,432,433,434,435,440,441,443,44 >>>>>>>>>> 5, >>>>>>>>>> 44 >>>>>>>>>> 7, >>>>>>>>>>>> >>>>>>>>>>>> >>>>>>>>>> 4 >>>>>>>>>>>> >>>>>>>>>> 49,450,452,454,455,456,461,464,466,470,472,473,481,487,488,491,492,49 >>>>>>>>>> 3, >>>>>>>>>> 49 >>>>>>>>>> 4, >>>>>>>>>>>> >>>>>>>>>>>> >>>>>>>>>> 4 >>>>>>>>>>>> 95,496,497,498,499,501,502,504,506,507,509,511,513,515,516,51 [... >>>>>>>>>>>> truncated] >>>>>>>>>>>> >>>>>>>>>>>> What exactly is 'mydata', and how did you generate it? The above >>>>>>>>>>>> error >>>>>>>>>>>> indicates that you have duplicate row names, which IIRC isn't >>>>>>>>>>>> possible >>>>>>>>>>>> to do with an expressionSet. >>>>>>>>>>>> >>>>>>>>>>>> R(9062,0xa05c5540) malloc: *** mmap(size=458665984) failed (error >>>>>>>>>>>> code=12) >>>>>>>>>>>> *** error: can't allocate region >>>>>>>>>>>> *** set a breakpoint in malloc_error_break to debug >>>>>>>>>>>> R(9062,0xa05c5540) malloc: *** mmap(size=458665984) failed (error >>>>>>>>>>>> code=12) >>>>>>>>>>>> *** error: can't allocate region >>>>>>>>>>>> *** set a breakpoint in malloc_error_break to debug >>>>>>>>>>>> >>>>>>>>>>>> More lack of memory errors. >>>>>>>>>>>> >>>>>>>>>>>> >>>>>>>>>>>> Error in help(dt[i], package = pkg[i], htmlhelp = TRUE) : >>>>>>>>>>>> unused argument(s) (htmlhelp = TRUE) >>>>>>>>>>>> In addition: Warning messages: >>>>>>>>>>>> 1: In data(package = .packages(all.available = TRUE)) : >>>>>>>>>>>> datasets have been moved from package 'base' to package >>>>>>>>>>>> 'datasets' >>>>>>>>>>>> 2: In data(package = .packages(all.available = TRUE)) : >>>>>>>>>>>> datasets have been moved from package 'stats' to package >>>>>>>>>>>> 'datasets' >>>>>>>>>>>> starting httpd help server ... done >>>>>>>>>>>> >>>>>>>>>>>> Would someone be able to diagnose the problem and suggest a >>>>>>>>>>>> solution? >>>>>>>>>>>> >>>>>>>>>>>> First, get more RAM. Second, you will be better off using a 64-bit >>>>>>>>>>>> OS. >>>>>>>>>>>> Depending on your hardware, you might be able to just install a >>>>>>>>>>>> 64-bit >>>>>>>>>>>> version of R. >>>>>>>>>>>> >>>>>>>>>>>> Best, >>>>>>>>>>>> >>>>>>>>>>>> Jim >>>>>>>>>>>> >>>>>>>>>>>> >>>>>>>>>>>> >>>>>>>>>>>> >>>>>>>>>>>> If it is useful, I am using the following R software: R for Mac OS >>>>>>>>>>>> X >>>>>>>>>>>> GUI >>>>>>>>>>>> 1.35-dev Leopard build 32-bit. If there is any other info that >>>>>>>>>>>> would >>>>>>>>>>>> be >>>>>>>>>>>> useful please let me know. >>>>>>>>>>>> >>>>>>>>>>>> I had a read of the AffyQCReport Package pdf and I have added the >>>>>>>>>>>> following >>>>>>>>>>>> line: QCReport(ReadAffy(widget=TRUE)). Then I tried >>>>>>>>>>>> library(affyQCReport); >>>>>>>>>>>> QCReport(mydata, file="ExampleQC.pdf") again. It now seems to be >>>>>>>>>>>> doing >>>>>>>>>>>> something, in other words it doesn?t go to the error, yet, but it?s >>>>>>>>>>>> been >>>>>>>>>>>> processing for about 10 minutes. I am analyzing 35 chips. >>>>>>>>>>>> >>>>>>>>>>>> Perhaps it would work if I tried to generate each QCReport page >>>>>>>>>>>> separately >>>>>>>>>>>> rather than as a whole. >>>>>>>>>>>> >>>>>>>>>>>> Cordially, >>>>>>>>>>>> Rick >>>>>>>>>>>> >>>>>>>>>>>> >>>>>>>>>>>> >>>>>>>>>>>> >>>>>>>>>>>> _______________________________________________ >>>>>>>>>>>> Bioconductor mailing list >>>>>>>>>>>> Bioconductor at r-project.org >>>>>>>>>>>> https://stat.ethz.ch/mailman/listinfo/bioconductor >>>>>>>>>>>> Search the archives: >>>>>>>>>>>> http://news.gmane.org/gmane.science.biology.informatics.conductor >>>>>>>>>>>> >>>>>>>>>> >>>>>>>> >>>>>> >>>> >> -- Rick Frausto PhD Candidate The University of Sydney School of Molecular Bioscience G08 Camperdown, NSW 2006 AUSTRALIA ricardo.frausto at sydney.edu.au Phone: 61 2 9036 5354 Lab of Iain L. Campbell