I see, Lukas. That makes sense. Thanks for explaining it. So, MEDIPS looks in the BSgenome object for seqnames only, and doesn't look in mseqnames. It seems like a lot of users of the MEDIPS package must encounter this problem, because most genomes have sets of unassembled scaffolds that, following the BSgenome forging instructions, inevitably end up as mseq objects. It would be great if a future version of MEDIPS looked for both types of sequences in the BSgenome. I also meant to reply to Steve's question about this line from the vignette: BSgenome="BSgenome.Hsapiens.UCSC.hg19" In my case, with my custom genome, this would be: BSgenome = "BSgenome.Ptrichocarpa.Phytozome.v3" But when I do this, the literal BSgenome.Ptrichocarpa.Phytozome.v3 is assigned to the BSgenome variable as a character vector. So instead, I do this: BSgenome = BSgenome.Ptrichocarpa.Phytozome.v3 That works. So maybe quotes are only required for genomes listed under available_genomes()? Meanwhile, for my MEDIPS analysis, I'm willing to go back to the bam files and filter out the scaffold alignments. It looks like Rsamtools has methods for this. Should I be trying to set up a FilterRules instance, or a ScanBamParam instance? Any help with the syntax for this step will be appreciated. I'm sure this is not going to work, but I'll start with something like this: bamFilt_Fall1 <- BamFile(filterBam(file, filter=FilterRules("scaffold"))) Thanks much, --Kelly From: Lukas Chavez [mailto:lukas.chavez.mailings@googlemail.com] Sent: Tuesday, April 01, 2014 12:32 AM To: Vining, Kelly Cc: Steve Lianoglou; bioconductor@r-project.org Subject: Re: [BioC] MEDIPS.createSet error Thank you Steve for explaining in detail how to look at the content of the BSgenome object and the bam file. When MEDIPS imports the bam file, it will try to extract information for the 'scaffold_' chromosomes from the BSgenome object. The initially reported error Calculating genomic coordinates...Error in vector(length = supersize_chr[length( chromosomes)], mode = "character") : vector size cannot be NA/NaN occurs because MEDIPS is trying to find the length of a chromosome given in the bam file, but does not find the chromosome in the BSgenome object. Subsequently, MEDIPS tries to calculate genomic coordinates for the chromosome wide windows, but the length of the chromosome is NA. I understand that it will be necessary to catch this error and add a warning message in a future version of MEDIPS. If you want to keep your hits on the 'scaffold_' chromosomes in the bam file, and to include all scaffolds in your further analysis, you will have to recreate your BSgenome object treating each scaffold as an individual chromosome just as the other assembled chromosomes. Lukas On Mon, Mar 31, 2014 at 3:05 PM, Vining, Kelly <kelly.vining@oregonstate.edu<mailto:kelly.vining@oregonstate.edu>> wrote: Thanks, Steve, for your advice - appreciate you working with a package that isn't familiar to you. Thanks also for catching that I didn't have Rsamtools installed. I think this output that you suggested I generate gives an idea about what might be going on: > si.bsg Seqinfo of length 19 seqnames seqlengths isCircular genome Chr01 50495391 FALSE 3.0 Chr02 25263035 FALSE 3.0 Chr03 21816808 FALSE 3.0 Chr04 24267051 FALSE 3.0 Chr05 25890704 FALSE 3.0 ... ... ... ... Chr15 15278577 FALSE 3.0 Chr16 14494361 FALSE 3.0 Chr17 16080358 FALSE 3.0 Chr18 16958300 FALSE 3.0 Chr19 15942145 FALSE 3.0 > si.bam Seqinfo of length 1446 seqnames seqlengths isCircular genome Chr01 50495391 <na> <na> Chr02 25263035 <na> <na> Chr03 21816808 <na> <na> Chr04 24267051 <na> <na> Chr05 25890704 <na> <na> ... ... ... ... scaffold_3584 3654 <na> <na> scaffold_3595 2008 <na> <na> scaffold_3648 2796 <na> <na> scaffold_3664 3053 <na> <na> scaffold_3681 1022 <na> <na> When I created the reference genome structure, the scaffolds were all entered in a single, multiple seq object, following instructions. But in my bam files, the scaffold hits are as shown above. So that could be one problem, but I don't know how to solve that other than filtering out all of the alignments to the scaffolds from the bam files. I really would like to retain the scaffold alignments and not lose all of that precious data. The other potential issue is the "NA" entries in the "isCircular" and "genome" columns. I'm not sure whether those would be a problem. Continued thanks, --Kelly ________________________________________ From: Steve Lianoglou [lianoglou.steve@gene.com<mailto:lianoglou.steve@gene.com>] Sent: Monday, March 31, 2014 1:15 PM To: Vining, Kelly Cc: Lukas Chavez; bioconductor@r-project.org<mailto:bioconductor@r-project.org> Subject: Re: [BioC] MEDIPS.createSet error Hi, Caveat being that I've never used MEDIPS, and I'm just going along with the vignette. So, comments inline: On 31 Mar 2014, at 13:09, Vining, Kelly wrote: > Hi, > Thanks for the advice thus far! To confirm what is in my BSgenome > variable, I did this: > >> class(BSgenome) > [1] "BSgenome" > attr(,"package") > [1] "BSgenome" >> names(BSgenome) > [1] "Chr01" "Chr02" "Chr03" "Chr04" "Chr05" "Chr06" "Chr07" "Chr08" > "Chr09" > [10] "Chr10" "Chr11" "Chr12" "Chr13" "Chr14" "Chr15" "Chr16" "Chr17" > "Chr18" > [19] "Chr19" "scaf" > > And then: >> print(BSgenome) > Black cottonwood genome > | > | organism: Populus trichocarpa (Black cottonwood) > | provider: Phytozome (JGI) > | provider version: 3.0 > | release date: January 2010 > | release name: Populus trichocarpa v3.0 > | > | single sequences (see '?seqnames'): > | Chr01 Chr02 Chr03 Chr04 Chr05 Chr06 Chr07 Chr08 Chr09 > Chr10 Chr11 > | Chr12 Chr13 Chr14 Chr15 Chr16 Chr17 Chr18 Chr19 > | > | multiple sequences (see '?mseqnames'): > | scaf > | > | (use the '$' or '[[' operator to access a given sequence) > > > So that looks ok. Interestingly, when I followed the vignette and did > the equivalent of > BSgenome="BSgenome.Hsapiens.UCSC.hg19" The vignette suggests that BSgenome should be the package name of the BSgenome package to open, so yours should be something like "BSgenome.Ptrichocarpa.XXX.YY" or something -- I guess you built this packge by yourself, or something, so you'd know its name ... > That didn't work for me. It only worked without quotes. If I included > quotes, it just assigned a character vector to that variable. Sorry, what exactly didn't work for you? Can you show me the code that failed? > Then, following your advice: > >> si.bsg <- seqinfo(BSgenome.Ptrichocarpa.Phytozome.v3) >> si.bam <- seqinfo(BamFile("FallBud_brep1_aln_sorted.bam")) > Error in seqinfo(BamFile("FallBud_brep1_aln_sorted.bam")) : > error in evaluating the argument 'x' in selecting a method for > function 'seqinfo': Error: could not find function "BamFile" The BamFile function is defined in the Rsamtools package, you need to load that first (the first line of code I suggested you run was to load the Rsamtools package). Load it first, then redo the seqinfo(BamFile(...)) stuff. -steve [[alternative HTML version deleted]]