Hi Dan, Thanks for your suggestion. I did look at the pasilla package before I emailed the list, but it doesn't seem to contain bam files: ---- lsa-579-005:~ darwin$ ls -l /Library/Frameworks/R.framework/Versions/2 .15/Resources/library/pasilla/extdata total 46184 -rw-r--r-- 1 darwin admin 13589253 Oct 2 13:04 Dmel.BDGP5.25.62.DEXSeq.chr.gff -rw-r--r-- 1 darwin admin 552 Oct 2 13:04 geneIDsinsubset.txt -rw-r--r-- 1 darwin admin 498373 Oct 2 13:04 pasilla_gene_counts.tsv -rw-r--r-- 1 darwin admin 1370030 Oct 2 13:04 treated1fb.txt -rw-r--r-- 1 darwin admin 1359480 Oct 2 13:04 treated2fb.txt -rw-r--r-- 1 darwin admin 1361730 Oct 2 13:04 treated3fb.txt -rw-r--r-- 1 darwin admin 1363665 Oct 2 13:04 untreated1fb.txt -rw-r--r-- 1 darwin admin 1371149 Oct 2 13:04 untreated2fb.txt -rw-r--r-- 1 darwin admin 1358790 Oct 2 13:04 untreated3fb.txt -rw-r--r-- 1 darwin admin 1359414 Oct 2 13:04 untreated4fb.txt ----- lsa-579-005:~ darwin$ head /Library/Frameworks/R.framework/Versions/2. 15/Resources/library/pasilla/extdata/treated1fb.txt FBgn0000003:001 0 FBgn0000008:001 0 FBgn0000008:002 0 FBgn0000008:003 0 FBgn0000008:004 1 FBgn0000008:005 4 FBgn0000008:006 1 FBgn0000008:007 18 FBgn0000008:008 4 FBgn0000008:009 16 Or are those '*fb.txt' files some sort of "debinaried" bam files? Again, thanks for your suggestion. Best wishes, Darwin On Jan 10, 2013, at 9:38 PM, Dan Tenenbaum wrote: > On Thu, Jan 10, 2013 at 9:26 PM, Vincent Carey > <stvjc at="" channing.harvard.edu=""> wrote: >> The original poster seems to be referring to a line in the pasilla vignette >> >> The SAM alignment files from which \Rpackage{pasilla} was generated are >> available at \url{http://www.embl.de/~reyes/Graveley/bam}. >> >> and indeed it seems this link is dead. but it doesn't really matter for >> the vignette as the >> pasillaBamSubset package has the bam files needed. >> > > You might also look at the pasilla package. > Dan > > >> On Fri, Jan 11, 2013 at 12:13 AM, Paul Shannon <pshannon at="" fhcrc.org=""> wrote: >> >>> Hi Darwin, >>> >>> Please give us a little bit more detail on the tutorial you wish to >>> follow. Is it one of the vignettes that accompany GenomicRanges? >>> >>> Also, your sessionInfo() may provide indispensable clues allowing us to >>> help out. >>> >>> Thanks - >>> >>> - Paul >>> >>> On Jan 10, 2013, at 8:33 PM, Darwin Sorento Dichmann wrote: >>> >>>> Greetings, >>>> >>>> I wish to follow the tutorial for summarizeOverlaps from GenomicRanges, >>> but the pasilla.bam files ("treated1.bam", "untreated1.bam", >>> "untreated2.bam") are not with in the package and the provided link for >>> download is dead (http://www.embl.de/~reyes/Graveley/bam). >>>> >>>> Anybody know where I can get those data or have a copy? I also tried >>> following the GEO accessions from the original publication, but all I found >>> was GFFs and BEDs, no bams. >>>> >>>> Any help is greatly appreciated. >>>> >>>> Best, >>>> Darwin >>>> ________________________________ >>>> Darwin Sorento Dichmann, M.S., PhD >>>> University of California, Berkeley >>>> Harland Lab >>>> Molecular and Cell Biology >>>> 571 Life Sciences Addition >>>> Berkeley, CA 94720 >>>> Phone# (510) 643-7830 >>>> Fax# (510) 643-6791 >>>> E-mail: dichmann at berkeley.edu >>>> >>>> Please send Fedex packages to: >>>> 163 Life Sciences Addition, attn: Harland lab room 571 >>>> >>>> >>>> >>>> [[alternative HTML version deleted]] >>>> >>>> _______________________________________________ >>>> 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 >>> >>> _______________________________________________ >>> 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 >>> >> >> [[alternative HTML version deleted]] >> >> _______________________________________________ >> 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 > > _______________________________________________ > 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

Dear everyone, Just for the record, I have updated the link to the bam files in the pasilla vignette. The new url it should not die anymore. Bests, Alejandro > On Fri, Jan 11, 2013 at 9:13 AM, Martin Morgan <mtmorgan at="" fhcrc.org=""> wrote: > >> and Rsamtools::asBam >> > which is an awesomely simple and effective function, since it handles a > bunch of annoying SAM/BAM-related impedimentia by default! > > anyone who stumbles on a SAM file at some point will be happy to make its > acquaintance > thank you Herve and Martin for anticipating everyone else's common case :-) > >

On 01/11/2013 10:16 AM, Tim Triche, Jr. wrote: > On Fri, Jan 11, 2013 at 9:13 AM, Martin Morgan <mtmorgan at="" fhcrc.org=""> wrote: > >> and Rsamtools::asBam >> > > which is an awesomely simple and effective function, since it handles a > bunch of annoying SAM/BAM-related impedimentia by default! > > anyone who stumbles on a SAM file at some point will be happy to make its > acquaintance > thank you Herve and Martin Well, Martin in that case. Like 95% of the Rsamtools package. So thanks Martin! :-) H. > for anticipating everyone else's common case :-) > > -- Hervé Pagès Program in Computational Biology Division of Public Health Sciences Fred Hutchinson Cancer Research Center 1100 Fairview Ave. N, M1-B514 P.O. Box 19024 Seattle, WA 98109-1024 E-mail: hpages at fhcrc.org Phone: (206) 667-5791 Fax: (206) 667-1319

On 01/12/2013 09:50 AM, Valerie Obenchain wrote: > Hi Darwin, > > There are a couple of issues going on here. Single-end and paired- end > reads need to be handled separately. The BamFileList should hold either > single or paired-end and the 'singleEnd' argument needs to be specified. > See the ?BamFileList man page for examples. > > The output of summarizeOverlaps is a SummarizedExperiment object not a > countDataSet. To read more about this see the ?summarizeOverlaps man > page. In the summarizeOverlaps vignette, a countDataSet is created from > the counts in the assays() slot of the SummarizedExperiment object. > > More below. > > On 01/11/13 13:08, Darwin Sorento Dichmann wrote: >> Hi Valerie, >> >> Thanks for your response. I did try looking at the GEO location, but >> since the names of the 82 directories do not provide any information >> as to what files they contain (at least as far as I could tell), I did >> not proceed further with that:-P >> >> I did try the pasillaBamSubset yesterday before I emailed the list, >> but I had problems reading the files using BamFileList. I tried: >> >> --- >> > fls <- c("untreated1_chr4.bam", "untreated3_chr4.bam") >> > path <- >> "/Library/Frameworks/R.framework/Versions/2.15/Resources/library/pa sillaBamSubset/extdata/" >> >> > bamlst <- BamFileList(fls, index=character()) >> > genehits <- summarizeOverlaps(chr4genes, bamlst, mode="Union") >> Error in .io_check_exists(path(con)) : file(s) do not exist: >> 'untreated1_chr4.bam' >> --- >> I assume that it's because the path to the files (as stored in 'path') >> is not passed on to bamlst through BamFileList(?). The above was >> modified from the vignette, which reads: >> >> ---- >> > fls<- c("treated1.bam", "untreated1.bam", "untreated2.bam") >> > path<- "pathToBAMFiles" >> > bamlst<- BamFileList(fls, index=character()) >> > genehits<- summarizeOverlaps(chr4genes, bamlst, mode="Union") >> ---- >> I also tried supplying the full path to 'fls' like this: >> >> fls <- >> c("/Library/Frameworks/R.framework/Versions/2.15/Resources/library/ pasillaBamSubset/extdata/untreated1_chr4.bam", >> "/Library/Frameworks/R.framework/Versions/2.15/Resources/library/pa sillaBamSubset/extdata/untreated3_chr4.bam") >> > > A better way to do this is with system.file. See ?BamFileList for an > example. > > fl <- system.file("extdata", "untreated1_chr4.bam", > package="pasillaBamSubset", mustWork=TRUE) Or even better: untreated1_chr4() and untreated3_chr4() to get those paths. The main reason for putting those little wrappers in pasillaBamSubset was to make it easier to document the files: ?untreated1_chr4 (otherwise, and AFAIK, there is no built-in mechanism in R for documenting "external data"). H. > >> bamlst <- BamFileList(fls, index=character()) >> genehits <- summarizeOverlaps(chr4genes, bamlst, mode="Union") >> >> which works in the sense that the bams are read, but later yields a >> pretty messy 'design' matrix: >> > design >> condition replicate type >> countfiles >> 1 untreated 1 single-read >> /Library/Frameworks/R.framework/Versions/2.15/Resources/library/pas illaBamSubset/extdata/untreated1_chr4.bam >> >> 2 untreated 3 single-read >> /Library/Frameworks/R.framework/Versions/2.15/Resources/library/pas illaBamSubset/extdata/untreated3_chr4.bam >> > > This is correct. It is only 'messy' because the paths to the files are > long. > >> >> And lots of warnings when applying 'summarizeOverlaps': >> >> --- >> > genehits <- summarizeOverlaps(chr4genes, bamlst, mode="Union") >> Warning messages: >> 1: In .Seqinfo.mergexy(x, y) : >> Each of the 2 combined objects has sequence levels not in the other: >> - in 'x': chr2L, chr2R, chr3L, chr3R, chr4, chrM, chrX, chrYHet >> - in 'y': chrchr3R, chrchrX, chrchr4, chrchr3L, chrchr2LHet, >> chrchrU, chrchrXHet, chrchr2RHet, chrchrdmel_mitochondrion_genome, >> chrchrYHet, chrchr2R, chrchr3LHet, chrchr3RHet, chrchr2L >> Make sure to always combine/compare objects based on the same reference >> genome (use suppressWarnings() to suppress this warning). >> 2: In .Seqinfo.mergexy(x, y) : >> Each of the 2 combined objects has sequence levels not in the other: >> - in 'x': chr2L, chr2R, chr3L, chr3R, chr4, chrM, chrX, chrYHet >> - in 'y': chrchr3R, chrchrX, chrchr4, chrchr3L, chrchr2LHet, >> chrchrU, chrchrXHet, chrchr2RHet, chrchrdmel_mitochondrion_genome, >> chrchrYHet, chrchr2R, chrchr3LHet, chrchr3RHet, chrchr2L >> Make sure to always combine/compare objects based on the same reference >> genome (use suppressWarnings() to suppress this warning). >> --- > > These warnings indicate mismatches between your annotation (chr4genes) > and your Bam file (bamlst). > > - in 'x': chr2L, chr2R, chr3L, chr3R, chr4, chrM, chrX, chrYHet > > - in 'y': chrchr3R, chrchrX, chrchr4, chrchr3L, chrchr2LHet, chrchrU, > chrchrXHet, ... > > Your chromosome names do not match. Look at the example on the ?BamFile > man page in the 'summarizeOverlaps with BamFileList' section and make > sure you can run that. Some important things to note: > > - The output of summarizeOverlaps is a SummarizedExperiment > - A countDataSet can be created from the counts in the assays() slot of > the SummarizedExperiment object > - untreated1 and untreated3 are treated separately because they are > single- and paired-end > - yieldSize can be used to iterate through large files > > > Valerie > >> >> From what I understand the end results should be a 'CountDataSet' >> object for use in further downstream analysis, and it looks like I do >> end up with that: >> --- >> > geneCDS >> CountDataSet (storageMode: environment) >> assayData: 82 features, 2 samples >> element names: counts >> protocolData: none >> phenoData >> sampleNames: >> /Library/Frameworks/R.framework/Versions/2.15/Resources/library/pas illaBamSubset/extdata/untreated1_chr4.bam >> >> /Library/Frameworks/R.framework/Versions/2.15/Resources/library/pas illaBamSubset/extdata/untreated3_chr4.bam >> >> varLabels: sizeFactor condition ... countfiles (5 total) >> varMetadata: labelDescription >> featureData: none >> experimentData: use 'experimentData(object)' >> pubMedIds: 20921232 >> Annotation: >> --- >> I'll look into how to query CountDataSets to see if it really worked. >> >> I would appreciate if someone could help me understand how to supply >> the path to bam files to BamFileList in a smarter way than I did >> above. I look forward to applying these great tools. >> >> And thank you everybody for your suggestions! >> >> Best wishes, >> Darwin >> >> >> On Jan 11, 2013, at 9:09 AM, Valerie Obenchain wrote: >> >>> Hi Darwin, >>> >>> As Vince mentioned, the bam files are no longer available at the >>> location specified in the summarizeOverlaps vignette. This location >>> was taken from the DEXSeq vignette which has since been updtated to >>> point to the GEO location, >>> >>> http://www.ncbi.nlm.nih.gov/projects/geo/query/acc.cgi?acc=GSE18508 >>> >>> Available file types include GFF, SAM and BEDGRAPH. The SAM can be >>> easily converted to BAM with samtools >>> >>> samtools view -h -o outputFile.sam inputFile.bam >>> >>> >>> As an fyi, we have a Bioconductor data package 'pasillaBamSubset' >>> which includes a portion of chromosome 4 from the untreated1 >>> (single-end) and untreated3 (paired-end) files. You may find these >>> smaller files useful for testing. >>> >>> Thanks for the reminder of the dead link. I will update the vignette. >>> >>> >>> Valerie >>> >>> >>> >>> On 01/10/2013 08:33 PM, Darwin Sorento Dichmann wrote: >>>> Greetings, >>>> >>>> I wish to follow the tutorial for summarizeOverlaps from >>>> GenomicRanges, but the pasilla.bam files ("treated1.bam", >>>> "untreated1.bam", "untreated2.bam") are not with in the package and >>>> the provided link for download is dead >>>> (http://www.embl.de/~reyes/Graveley/bam). >>>> >>>> Anybody know where I can get those data or have a copy? I also tried >>>> following the GEO accessions from the original publication, but all >>>> I found was GFFs and BEDs, no bams. >>>> >>>> Any help is greatly appreciated. >>>> >>>> Best, >>>> Darwin >>>> ________________________________ >>>> Darwin Sorento Dichmann, M.S., PhD >>>> University of California, Berkeley >>>> Harland Lab >>>> Molecular and Cell Biology >>>> 571 Life Sciences Addition >>>> Berkeley, CA 94720 >>>> Phone# (510) 643-7830 >>>> Fax# (510) 643-6791 >>>> E-mail: dichmann at berkeley.edu >>>> >>>> Please send Fedex packages to: >>>> 163 Life Sciences Addition, attn: Harland lab room 571 >>>> >>>> >>>> >>>> [[alternative HTML version deleted]] >>>> >>>> _______________________________________________ >>>> 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 >>> >> > > _______________________________________________ > 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 -- Hervé Pagès Program in Computational Biology Division of Public Health Sciences Fred Hutchinson Cancer Research Center 1100 Fairview Ave. N, M1-B514 P.O. Box 19024 Seattle, WA 98109-1024 E-mail: hpages at fhcrc.org Phone: (206) 667-5791 Fax: (206) 667-1319