Thank you very much for your quick answers and looking in detail into this as well as updating the package. The updateObject(OBJECT) function works perfectly ! :-) Yes the corrected design matrix makes sense. I was really exploring and did not fully understand how blockfinder worked yet. Will look at the bumphunter function as well. Looking forward to the more detailed example :-) Thanks again, Florence 2013/10/24 Kasper Daniel Hansen <kasperdanielhansen@gmail.com> > Btw, thank you for an extremely useful example/bug report. > > Best, > Kasper > > > On Wed, Oct 23, 2013 at 11:59 PM, Kasper Daniel Hansen < > kasperdanielhansen@gmail.com> wrote: > >> Here is a list of the reported issues >> 1) The object returned by mapToGenome has is.unsorted return TRUE. This >> is now fixed, see NEWS below. >> 2) When bumphunter() returns zero bumps, it throws an error. On the todo >> list. >> 3) Is there an issue with the renaming of Relation_to_UCSC_CpG_Island to >> Relation_to_Island in cpgCollapse and helper function? The answer is yes, >> it was a bug and it is fixed now. >> >> Re. your example, your design matrix for the block finding is wrong. You >> are testing whether samples in group B has a methylation of 0.5, which is >> obviously not true. This is why your inferred cutoff is 12033998108438140 >> - you're easily off by a factor 10^15 (!!!!). You need to remove the 0+ in >> the model matrix line, and just have design = model.matrix(~+factor()). In >> fact, even better is >> design = model.matrix(~ gmSet$Sample_Group) >> >> Finally, we are working on a more detailed example for the vignette, >> but it is not done yet. >> >> Fixes are in minfi 1.8.3 (1.9.3 devel) which should be available from the >> build system in around 36h. >> >> * The function mapToGenome would return something that looked >> like an unordered GenomicMethylSet. Actually, loci were >> correctly ordered within chromosomes, the issue had to do >> with whether the chromosomes were ordered as chr1, chr2, chr3 >> (used in minfi) or chr1, chr10, chr11 (lexigraphically). >> Reported by Florence Cavalli <florence@ebi.ac.uk>. >> >> * Bug in cpgCollapse led to incorrect results. Your output is >> affected if table(granges(output[[1]])$type) is all >> 'OpenSea'. Reported by Florence Cavalli >> <florence@ebi.ac.uk>. >> >> Best, >> Kasper >> >> >> >> >> >> On Tue, Oct 22, 2013 at 8:27 PM, Kasper Daniel Hansen < >> kasperdanielhansen@gmail.com> wrote: >> >>> 1) You can update your objects by running >>> OBJECT = updateObject(OBJECT) >>> This fixes everything and there should be no problems. Unless you have >>> been using the hg18 coordinates from the old annotation package, in which >>> case you're out of luck. >>> >>> The reason for this is that I realized that a substantial amount of the >>> annotation material provided by illumina of course depends on the genome >>> build, for example relation to CpG island. And Illumina only provides this >>> for hg19, not hg18. >>> >>> 2) A lot of the other errors you have seems to be real errors probably >>> caused by some late re-organization of the code. Thanks for providing an >>> extremely nice example where they fail, which should make it very easy to >>> track down. I suspect some late code cleaning errors to be behind this. I >>> will investigate and report back very soon. >>> >>> 3) If you want to find DMRs, which we define as smaller regions like >>> 100bp-5kb or so, you want to use >>> bumphunter() >>> The function blockFinder is for finding "blocks" which are large scale >>> DMRs, on the megabase scale, like in our 'recent' Nat Genet paper. Not >>> sure if that is what you want? >>> >>> Best, >>> Kasper >>> >>> >>> On Tue, Oct 22, 2013 at 4:04 PM, Florence Cavalli <florence@ebi.ac.uk>wrote: >>> >>>> Hello, >>>> >>>> I am working with 450k methylation data. >>>> - Is there a way to modify the annotation linked to an MethylSet object, >>>> (or is this something we are not supposed to do)? >>>> I created my large MethylSet object under the pervious version of R and >>>> Bioc and at the time the annotation package >>>> IlluminaHumanMethylation450kannotation.ilmn.v1.2 was used. However this >>>> package is not available in BioC 2.13 >>>> but IlluminaHumanMethylation450kanno.ilmn12.hg19 replaced it (as far as >>>> I >>>> understood). >>>> >>>> I would like to be able to do this since I'd like to use the >>>> new cpgCollapse() and blockFinder() functions from minfi that are under >>>> BioC 2.13. Indeed cpgCollapse() calls getAnnotation() and since I cannot >>>> load the annotation package in R-3.0.2/BioC 2.13 an error is returned >>>> (and >>>> the new functions are not available under the previous version of BioC). >>>> >>>> Is there a way around this problem ? I tried the annotation() function >>>> but >>>> without success. Or do I have to reconstruct my MethylSet object from >>>> the >>>> very beginning reading the raw data using the library >>>> IlluminaHumanMethylation450kanno.ilmn12.hg19, normalysing (and >>>> re-running >>>> my clustering to be sure to have consistent results) ? It is just that I >>>> would save quite some time if I could actually convert my object. >>>> >>>> - Also I am planning to use the the following workflow to run >>>> blockFinder() >>>> and find differentially methylated regions. >>>> >>>> To be able to ultimately run blockFinder(), using your the minfi test >>>> data >>>> I created a GenomicMethylSet with mapToGenome() function from >>>> the MethylSet object then I ran cpgCollapse() to obtain an >>>> GenomicRatioSet object and finally used this object and a design >>>> matrix to >>>> run blockFinder(). >>>> See example: >>>> >>>> As in the vignette: >>>> library(IlluminaHumanMethylation450kmanifest) >>>> library(IlluminaHumanMethylation450kanno.ilmn12.hg19) >>>> library(minfi) >>>> require(minfiData) >>>> >>>> baseDir <- system.file("extdata", package = "minfiData") >>>> targets <- read.450k.sheet(baseDir) >>>> targets >>>> >>>> sub(baseDir, "", targets$Basename) >>>> RGset <- read.450k.exp(base = baseDir, targets = targets) >>>> RGset >>>> pd <- pData(RGset) >>>> pd[,1:4] >>>> >>>> Then >>>> >>>> MSet.norm <- preprocessIllumina(RGset, bg.correct = TRUE, normalize = >>>> "controls") >>>> Mset.swan <- preprocessSWAN(RGset, MSet.norm) >>>> >>>> mset <- Mset.swan >>>> >>>> >>>> > mset >>>> MethylSet (storageMode: lockedEnvironment) >>>> assayData: 485512 features, 6 samples >>>> element names: Meth, Unmeth >>>> phenoData >>>> sampleNames: 5723646052_R02C02 5723646052_R04C01 ... >>>> 5723646053_R06C02 (6 total) >>>> varLabels: Sample_Name Sample_Well ... filenames (13 total) >>>> varMetadata: labelDescription >>>> Annotation >>>> array: IlluminaHumanMethylation450k >>>> annotation: ilmn12.hg19 >>>> Preprocessing >>>> Method: SWAN (based on a MethylSet preprocesses as 'Illumina, >>>> bg.correct >>>> = TRUE, normalize = controls, reference = 1' >>>> minfi version: 1.8.1 >>>> Manifest version: 0.4.0 >>>> > >>>> >>>> > class(mset) >>>> [1] "MethylSet" >>>> attr(,"package") >>>> [1] "minfi" >>>> > gmSet = mapToGenome(mset) >>>> > class(gmSet) >>>> [1] "GenomicMethylSet" >>>> attr(,"package") >>>> [1] "minfi" >>>> > gmSet_cpgCollapse=cpgCollapse(gmSet,what="M",returnBlockInfo=FALSE) >>>> [cpgCollapse] Creating annotation. >>>> Error in cpgCollapseAnnotation(gr, relationToIsland, islandName, maxGap >>>> = >>>> maxGap, : >>>> object has to be ordered. >>>> >>>> Not sure about the following !? But I seems that sorting the object >>>> works >>>> > gmSet_s=sort(gmSet) >>>> > gmSet_cpgCollapse=cpgCollapse(gmSet_s,what="M",returnBlockInfo=FALSE) >>>> [cpgCollapse] Creating annotation. >>>> [cpgCollapseAnnotation] Clustering islands and clusters of probes. >>>> [cpgCollapseAnnotation] Computing new annotation. >>>> [cpgCollapseAnnotation] Defining blocks. >>>> [cpgCollapse] Collapsing data...... >>>> > class(gmSet_cpgCollapse) >>>> [1] "GenomicRatioSet" >>>> attr(,"package") >>>> [1] "minfi" >>>> >>>> ## Sample groups >>>> ## > targets[,"Sample_Group"] >>>> ## [1] "GroupA" "GroupA" "GroupB" "GroupB" "GroupA" "GroupB" >>>> > design <- model.matrix(~ 0+factor(c(1,1,2,2,1,2))) >>>> > colnames(design) <- c("groupA", "groupB") >>>> > head(design) >>>> groupA groupB >>>> 1 1 0 >>>> 2 1 0 >>>> 3 0 1 >>>> 4 0 1 >>>> 5 1 0 >>>> 6 0 1 >>>> >>>> > Block_g2=blockFinder(gmSet_cpgCollapse, design=design, coef = 2, what >>>> = >>>> "M",pickCutoff=TRUE,smooth=FALSE) >>>> [bumphunterEngine] Using a single core (backend: doSEQ, version: 1.4.1). >>>> [bumphunterEngine] Computing coefficients. >>>> [bumphunterEngine] Performing 1000 permutations. >>>> [bumphunterEngine] Computing marginal permutation p-values. >>>> [bumphunterEngine] cutoff: 12033998108438140 >>>> [bumphunterEngine] Finding regions. >>>> [bumphunterEngine] No bumps found! >>>> Error in res$table$indexStart : $ operator is invalid for atomic vectors >>>> >>>> I assume that if some bumps are found it would not return an error!? >>>> Would you advice to smooth the signal or not? >>>> >>>> Is that the proper way of running this analysis ? (I wonder since I did >>>> not >>>> find any example for such analysis) >>>> >>>> - Finally I noticed that in the cpgCollapse function the attribute >>>> Relation_to_UCSC_CpG_Island and UCSC_CpG_Islands_Name from a data frame >>>> return by getAnnotation object are called. >>>> However looking at the result of the getAnnotation() function called >>>> there >>>> isn't any Relation_to_UCSC_CpG_Island and UCSC_CpG_Islands_Name columns >>>> attributes (see below)? Are we supposed to use another annotation >>>> package? >>>> >>>> >>>> > cpgCollapse >>>> function (object, what = c("Beta", "M"), maxGap = 500, blockMaxGap = >>>> 2.5 * >>>> 10^5, maxClusterWidth = 1500, dataSummary = colMeans, na.rm = FALSE, >>>> returnBlockInfo = TRUE, verbose = TRUE, ...) >>>> { >>>> what <- match.arg(what) >>>> gr <- granges(object) >>>> ann <- getAnnotation(object) >>>> relationToIsland <- ann$Relation_to_UCSC_CpG_Island >>>> islandName <- ann$UCSC_CpG_Islands_Name >>>> .... >>>> .... >>>> } >>>> >>>> class(gmSet) >>>> gmSet >>>> > class(gmSet) >>>> [1] "GenomicMethylSet" >>>> attr(,"package") >>>> [1] "minfi" >>>> > gmSet >>>> class: GenomicMethylSet >>>> dim: 485512 6 >>>> exptData(0): >>>> assays(2): Meth Unmeth >>>> rownames(485512): cg13869341 cg14008030 ... cg08265308 cg14273923 >>>> rowData metadata column names(0): >>>> colnames(6): 5723646052_R02C02 5723646052_R04C01 ... 5723646053_R05C02 >>>> 5723646053_R06C02 >>>> colData names(13): Sample_Name Sample_Well ... Basename filenames >>>> Annotation >>>> array: IlluminaHumanMethylation450k >>>> annotation: ilmn12.hg19 >>>> Preprocessing >>>> Method: SWAN (based on a MethylSet preprocesses as 'Illumina, >>>> bg.correct >>>> = TRUE, normalize = controls, reference = 1' >>>> minfi version: 1.8.1 >>>> Manifest version: 0.4.0 >>>> >>>> >>>> A=getAnnotation(gmSet) >>>> colnames(A) >>>> >>>> > A=getAnnotation(gmSet) >>>> > colnames(A) >>>> [1] "chr" "pos" >>>> [3] "strand" "Name" >>>> [5] "AddressA" "AddressB" >>>> [7] "ProbeSeqA" "ProbeSeqB" >>>> [9] "Type" "NextBase" >>>> [11] "Color" "Probe_rs" >>>> [13] "Probe_maf" "CpG_rs" >>>> [15] "CpG_maf" "SBE_rs" >>>> [17] "SBE_maf" "Islands_Name" >>>> [19] "Relation_to_Island" "Forward_Sequence" >>>> [21] "SourceSeq" "Random_Loci" >>>> [23] "Methyl27_Loci" "UCSC_RefGene_Name" >>>> [25] "UCSC_RefGene_Accession" "UCSC_RefGene_Group" >>>> [27] "Phantom" "DMR" >>>> [29] "Enhancer" "HMM_Island" >>>> [31] "Regulatory_Feature_Name" "Regulatory_Feature_Group" >>>> [33] "DHS" >>>> > A$Relation_to_UCSC_CpG_Island >>>> NULL >>>> > A$UCSC_CpG_Islands_Name >>>> NULL >>>> >>>> sessionInfo() >>>> R version 3.0.2 (2013-09-25) >>>> Platform: x86_64-unknown-linux-gnu (64-bit) >>>> >>>> locale: >>>> [1] LC_CTYPE=en_US.UTF-8 LC_NUMERIC=C >>>> [3] LC_TIME=en_US.UTF-8 LC_COLLATE=en_US.UTF-8 >>>> [5] LC_MONETARY=en_US.UTF-8 LC_MESSAGES=en_US.UTF-8 >>>> [7] LC_PAPER=en_US.UTF-8 LC_NAME=C >>>> [9] LC_ADDRESS=C LC_TELEPHONE=C >>>> [11] LC_MEASUREMENT=en_US.UTF-8 LC_IDENTIFICATION=C >>>> >>>> attached base packages: >>>> [1] parallel stats graphics grDevices utils datasets methods >>>> [8] base >>>> >>>> other attached packages: >>>> [1] minfiData_0.4.2 >>>> [2] BiocInstaller_1.12.0 >>>> [3] IlluminaHumanMethylation450kanno.ilmn12.hg19_0.2.0 >>>> [4] IlluminaHumanMethylation450kmanifest_0.4.0 >>>> [5] minfi_1.8.1 >>>> [6] bumphunter_1.2.0 >>>> [7] locfit_1.5-9.1 >>>> [8] iterators_1.0.6 >>>> [9] foreach_1.4.1 >>>> [10] Biostrings_2.30.0 >>>> [11] GenomicRanges_1.14.1 >>>> [12] XVector_0.2.0 >>>> [13] IRanges_1.20.0 >>>> [14] reshape_0.8.4 >>>> [15] plyr_1.8 >>>> [16] lattice_0.20-24 >>>> [17] Biobase_2.22.0 >>>> [18] BiocGenerics_0.8.0 >>>> >>>> loaded via a namespace (and not attached): >>>> [1] annotate_1.40.0 AnnotationDbi_1.24.0 base64_1.1 >>>> [4] beanplot_1.1 codetools_0.2-8 DBI_0.2-7 >>>> [7] digest_0.6.3 doRNG_1.5.5 genefilter_1.44.0 >>>> [10] grid_3.0.2 illuminaio_0.4.0 itertools_0.1-1 >>>> [13] limma_3.18.0 MASS_7.3-29 matrixStats_0.8.12 >>>> [16] mclust_4.2 multtest_2.18.0 nlme_3.1-111 >>>> [19] nor1mix_1.1-4 pkgmaker_0.17.4 preprocessCore_1.24.0 >>>> [22] R.methodsS3_1.5.2 RColorBrewer_1.0-5 registry_0.2 >>>> [25] rngtools_1.2.3 RSQLite_0.11.4 siggenes_1.36.0 >>>> [28] splines_3.0.2 stats4_3.0.2 stringr_0.6.2 >>>> [31] survival_2.37-4 tools_3.0.2 XML_3.98-1.1 >>>> [34] xtable_1.7-1 >>>> >>>> >>>> Thank you very much for developing these very useful packages! >>>> Best, >>>> Florence >>>> >>>> -- >>>> Florence Cavalli, PhD. >>>> The Hospital for Sick Children >>>> Brain Tumour Research Centre >>>> 101 College Street, TMDT-11-701 >>>> Toronto, ON M5G1L7 >>>> Canada >>>> >>>> [[alternative HTML version deleted]] >>>> >>>> _______________________________________________ >>>> Bioconductor mailing list >>>> Bioconductor@r-project.org >>>> https://stat.ethz.ch/mailman/listinfo/bioconductor >>>> Search the archives: >>>> http://news.gmane.org/gmane.science.biology.informatics.conductor >>>> >>> >>> >> > [[alternative HTML version deleted]]