It works! RE: So close, but still error: RE: Rsamtools filtering bam file: RE: MEDIPS.createSet error
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Vining, Kelly ▴ 220
@vining-kelly-5029
Last seen 7.1 years ago
Hi Lukas, The first option appears to have worked! I have read in the first MEDIPS set. Following the tutuorial, it looks like additional biological reps get concatenated to the first one to make a list of MEDIPS sets. I'd like to read in all of my biological reps separately at first, though, because I have up to 5 reps for each of 3 treatments, and I ultimately want to be able to choose the three highest-quality reps for each treatment. I won't be able to tell which reps are highest quality until I run the quality control steps as outlined in the tutorial. One question about a detail of the MEDIPS.seqCoverage function: for the "pattern" parameter, does MEDIPS understand degenerate IUPAC nucleotide code, so that I could input, for example, "CHG"? If not, can I do something like "pattern=c(CAG,CTG,CCG)"? Or, do I have to use a brute force approach and get output for each pattern individually, then somehow merge all three outputs? Thanks again! --Kelly From: Lukas Chavez [mailto:lukas.chavez.mailings@googlemail.com] Sent: Wednesday, April 02, 2014 12:47 PM To: Vining, Kelly Cc: Martin Morgan; Steve Lianoglou; bioconductor@r-project.org Subject: Re: So close, but still error: RE: [BioC] Rsamtools filtering bam file: RE: MEDIPS.createSet error Dear Kelly, can you please use the following instead: bF1 = MEDIPS.createSet(file="FallBud_brep1_aln_sorted.bam", chr.select=seqnames(BSgenome.Ptrichocarpa.Phytozome.v3), BSgenome="BSgenome.Ptrichocarpa.Phytozome.v3", extend=300, uniq=TRUE, shift=0, window_size=1000) or BSgenome = "BSgenome.Ptrichocarpa.Phytozome.v3" chr.select = paste("Chr", formatC(seq(1:19), width=2, flag="0"), sep="") bF1 = MEDIPS.createSet(file="FallBud_brep1_aln_sorted.bam", chr.select=chr.select, BSgenome=BSgenome, extend=300, uniq=TRUE, shift=0, window_size=1000) and let me know if this works for you? Lukas On Wed, Apr 2, 2014 at 7:44 AM, Vining, Kelly <kelly.vining@oregonstate.edu<mailto:kelly.vining@oregonstate.edu>> wrote: Thanks Lukas, Martin and Steve for all of your help. MEDIPS.createSet almost works now. Alas, I'm still getting an error. Troubleshooting these error messages is giving me a deeper understanding of what is going on at each step, and that is good, but I am ready to get this working once and for all. Any help with this most recent error is much appreciated. --Kelly > bF1 = MEDIPS.createSet(file="FallBud_brep1_aln_sorted.bam", chr.select=seqnames(BSgenome), BSgenome="BSgenome", extend=300, uniq=TRUE, shift=0, window_size=1000) Reading bam alignment FallBud_brep1_aln_sorted.bam Selecting Chr01 Chr02 Chr03 Chr04 Chr05 Chr06 Chr07 Chr08 Chr09 Chr10 Chr11 Chr12 Chr13 Chr14 Chr15 Chr16 Chr17 Chr18 Chr19 Total number of imported short reads: 18306036 Extending reads... Creating GRange Object... Extract unique regions... Number of unique short reads: 15614381 Error in seqlengths(seqinfo(x)) : error in evaluating the argument 'x' in selecting a method for function 'seqlengths': Error in (function (classes, fdef, mtable) : unable to find an inherited method for function âseqinfoâ for signature â"function"â ________________________________ From: Lukas Chavez [lukas.chavez.mailings@googlemail.com<mailto:lukas. chavez.mailings@googlemail.com="">] Sent: Wednesday, April 02, 2014 1:03 AM To: Martin Morgan Cc: Vining, Kelly; Steve Lianoglou; bioconductor@r-project.org<mailto:bioconductor@r-project.org> Subject: Re: [BioC] Rsamtools filtering bam file: RE: MEDIPS.createSet error And yes, the argument BSgenome is supposed to be a character string naming the BSgenome object. Please let me know, if this will work for you. Lukas P.S. I like the chr.select=seqnames(BSgenome) idea- I think I will implement this as a default together with a warning/ info message. On Wed, Apr 2, 2014 at 12:46 AM, Lukas Chavez <lukas.chavez.mailings@g ooglemail.com<mailto:lukas.chavez.mailings@googlemail.com="">> wrote: Yes, the chr.select parameter will allow you to import selected chromosomes only from your bam files. I recommend to create an according bam index file (samtools) and to store it together with the bam file in the same directory. Lukas On Tue, Apr 1, 2014 at 1:49 PM, Martin Morgan <mtmorgan@fhcrc.org<mailto:mtmorgan@fhcrc.org>> wrote: On 04/01/2014 12:52 PM, Vining, Kelly wrote: This is a follow-up question about the most efficient way to use functions in Rsamtools to filter my bam files so that only alignments to chromosomes are included (extrachromosomal scaffold alignments are excluded). I think from your original question you are really looking to provide the names of the sequences in your BSgenome object as a value to the chr.select argument of MEDIPS.createSet, I *think* chr.select=seqnames(BSgenome) (be sure to only include seqnames defined in the bam file, too). >From the Rsamtools documentation, it looks like there are two ways to accomplish this: 1) bamFile(bamFilter()) ??? I'm not sure what you're talking about here, neither bamFile nor bamFilter are functions defined in Rsamtools ??? Use filterBam() to create a new bam file from an old bam files. It's use is illustrated in an example on the help page ?filterBam. 2) bamFile(ScanBamParam()) ScanBamParam() is a way to selectively input data, e.g., with the readGAlignments or scanBam functions. Its use is illustrated for instance on p. 2 of the vignette available in the Rsamtools package and at http://bioconductor.org/packages/release/bioc/vignettes/Rsamtools/inst /doc/Rsamtools-Overview.pdf MEDIPS.createSet uses a ScanBamParam internally, but as a user you do not have access to it. Martin For the first method, a "destination" output file name has to be designated. I don't want to create a separate file, so it appears that the second method is what I want, as it allows control of which records are imported and doesn't output a separate file. I set up a FilterRules object like so: filt <- FilterRules(list(isChr = function(x) rname(x) == "^Chr")) I also set up a "which" variable to contain the seqnames in my BSgenome: which <- seqnames(BSgenome) However, I'm getting stuck trying to apply either of these to ScanBamParam. Perhaps I want ScanBamWhat instead of ScanBamParam? Can someone clue me in about the proper syntax to accomplish this simple filtering operation? Thanks for any help, --Kelly ________________________________________ From: bioconductor-bounces@r-project.org<mailto:bioconductor- bounces@r-project.org=""> [bioconductor-bounces@r-project.org<mailto :bioconductor-bounces@r-project.org="">] on behalf of Vining, Kelly [Kelly.Vining@oregonstate.edu<mailto:kelly.vining@oregonstate.edu>] Sent: Tuesday, April 01, 2014 8:12 AM To: 'Lukas Chavez' Cc: Steve Lianoglou; bioconductor@r-project.org<mailto:bioconductor@r-project.org> Subject: Re: [BioC] MEDIPS.createSet error 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<mailto :lukas.chavez.mailings@googlemail.com="">] Sent: Tuesday, April 01, 2014 12:32 AM To: Vining, Kelly Cc: Steve Lianoglou; bioconductor@r-project.org<mailto: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@oregonsta te.edu<mailto:kelly.vining@oregonstate.edu=""><mailto:kelly.vining@oregon state.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=""><mailto: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-pro ject.org=""><mailto:bioconductor@r-project.org<mailto:bioconductor@r-proj ect.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]] _______________________________________________ Bioconductor mailing list Bioconductor@r-project.org<mailto:bioconductor@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@r-project.org<mailto:bioconductor@r-project.org> https://stat.ethz.ch/mailman/listinfo/bioconductor Search the archives: http://news.gmane.org/gmane.science.biology.informatics.conductor -- Computational Biology / Fred Hutchinson Cancer Research Center 1100 Fairview Ave. N. PO Box 19024 Seattle, WA 98109 Location: Arnold Building M1 B861 Phone: (206) 667-2793<tel:%28206%29%20667-2793> [[alternative HTML version deleted]] ADD COMMENT 0 Entering edit mode Lukas Chavez ▴ 570 @lukas-chavez-5781 Last seen 3.7 years ago USA/La Jolla/UCSD Dear Kelly, please excuse the delayed answer. MEDIPS.seqCoverage does not interpret IUPAC and accepts only one sequence pattern. Consequently, yes, you will need to run all possible combinations for CHG separately. Lukas MEDIPS.seqCoverage function: for the âpatternâ parameter, does MEDIPSunderstand degenerate IUPAC nucleotide code, so that I could input, for example, âCHGâ? If not, can I do something like âpattern=c(CAG,CTG,CCG)â? Or, do I have to use a brute force approach and get output for each pattern individually, then somehow merge all three outputs? On Thu, Apr 3, 2014 at 9:41 AM, Vining, Kelly <kelly.vining@oregonstate.edu>wrote: > Hi Lukas, > > The first option appears to have worked! I have read in the first MEDIPS > set. Following the tutuorial, it looks like additional biological reps get > concatenated to the first one to make a list of MEDIPS sets. Iâd like to > read in all of my biological reps separately at first, though, because I > have up to 5 reps for each of 3 treatments, and I ultimately want to be > able to choose the three highest-quality reps for each treatment. I wonât > be able to tell which reps are highest quality until I run the quality > control steps as outlined in the tutorial. > > > > One question about a detail of the MEDIPS.seqCoverage function: for the > âpatternâ parameter, does MEDIPS understand degenerate IUPAC nucleotide > code, so that I could input, for example, âCHGâ? If not, can I do something > like âpattern=c(CAG,CTG,CCG)â? Or, do I have to use a brute force approach > and get output for each pattern individually, then somehow merge all three > outputs? > > > > Thanks again! > > --Kelly > > > > *From:* Lukas Chavez [mailto:lukas.chavez.mailings@googlemail.com] > *Sent:* Wednesday, April 02, 2014 12:47 PM > *To:* Vining, Kelly > *Cc:* Martin Morgan; Steve Lianoglou; bioconductor@r-project.org > *Subject:* Re: So close, but still error: RE: [BioC] Rsamtools filtering > bam file: RE: MEDIPS.createSet error > > > > > Dear Kelly, > > can you please use the following instead: > > bF1 = MEDIPS.createSet(file="FallBud_brep1_aln_sorted.bam", > chr.select=seqnames(BSgenome.Ptrichocarpa.Phytozome.v3), > BSgenome="BSgenome.Ptrichocarpa.Phytozome.v3", extend=300, uniq=TRUE, > shift=0, window_size=1000) > > or > > BSgenome = "BSgenome.Ptrichocarpa.Phytozome.v3" > > chr.select = paste("Chr", formatC(seq(1:19), width=2, flag="0"), sep="") > bF1 = MEDIPS.createSet(file="FallBud_brep1_aln_sorted.bam", > chr.select=chr.select, BSgenome=BSgenome, extend=300, uniq=TRUE, shift=0, > window_size=1000) > > > > and let me know if this works for you? > > Lukas > > > > > > On Wed, Apr 2, 2014 at 7:44 AM, Vining, Kelly < > Kelly.Vining@oregonstate.edu> wrote: > > Thanks Lukas, Martin and Steve for all of your help. MEDIPS.createSet > almost works now. Alas, I'm still getting an error. Troubleshooting these > error messages is giving me a deeper understanding of what is going on at > each step, and that is good, but I am ready to get this working once and > for all. Any help with this most recent error is much appreciated. > > --Kelly > > > bF1 = MEDIPS.createSet(file="FallBud_brep1_aln_sorted.bam", > chr.select=seqnames(BSgenome), BSgenome="BSgenome", extend=300, uniq=TRUE, > shift=0, window_size=1000) > Reading bam alignment FallBud_brep1_aln_sorted.bam > Selecting Chr01 Chr02 Chr03 Chr04 Chr05 Chr06 Chr07 Chr08 Chr09 Chr10 > Chr11 Chr12 Chr13 Chr14 Chr15 Chr16 Chr17 Chr18 Chr19 > Total number of imported short reads: 18306036 > Extending reads... > Creating GRange Object... > Extract unique regions... > Number of unique short reads: 15614381 > Error in seqlengths(seqinfo(x)) : > error in evaluating the argument 'x' in selecting a method for function > 'seqlengths': Error in (function (classes, fdef, mtable) : > unable to find an inherited method for function Ã¢seqinfoÃ¢ for signature > Ã¢"function"Ã¢ > ------------------------------ > > *From:* Lukas Chavez [lukas.chavez.mailings@googlemail.com] > *Sent:* Wednesday, April 02, 2014 1:03 AM > *To:* Martin Morgan > *Cc:* Vining, Kelly; Steve Lianoglou; bioconductor@r-project.org > *Subject:* Re: [BioC] Rsamtools filtering bam file: RE: MEDIPS.createSet > error > > > And yes, the argument BSgenome is supposed to be a character string naming > the BSgenome object. Please let me know, if this will work for you. > > Lukas > > P.S. I like the chr.select=seqnames(BSgenome) idea- I think I will > implement this as a default together with a warning/ info message. > > > > On Wed, Apr 2, 2014 at 12:46 AM, Lukas Chavez < > lukas.chavez.mailings@googlemail.com> wrote: > > Yes, the chr.select parameter will allow you to import selected > chromosomes only from your bam files. I recommend to create an according > bam index file (samtools) and to store it together with the bam file in the > same directory. > > > Lukas > > > > On Tue, Apr 1, 2014 at 1:49 PM, Martin Morgan <mtmorgan@fhcrc.org> wrote: > > On 04/01/2014 12:52 PM, Vining, Kelly wrote: > > This is a follow-up question about the most efficient way to use functions > in Rsamtools to filter my bam files so that only alignments to chromosomes > are included (extrachromosomal scaffold alignments are excluded). > > > I think from your original question you are really looking to provide the > names of the sequences in your BSgenome object as a value to the chr.select > argument of MEDIPS.createSet, I *think* chr.select=seqnames(BSgenome) (be > sure to only include seqnames defined in the bam file, too). > > > > From the Rsamtools documentation, it looks like there are two ways to > accomplish this: > > 1) bamFile(bamFilter()) > > > ??? I'm not sure what you're talking about here, neither bamFile nor > bamFilter are functions defined in Rsamtools ??? > > Use filterBam() to create a new bam file from an old bam files. It's use > is illustrated in an example on the help page ?filterBam. > > 2) bamFile(ScanBamParam()) > > > ScanBamParam() is a way to selectively input data, e.g., with the > readGAlignments or scanBam functions. Its use is illustrated for instance > on p. 2 of the vignette available in the Rsamtools package and at > > > http://bioconductor.org/packages/release/bioc/vignettes/Rsamtools/in st/doc/Rsamtools-Overview.pdf > > MEDIPS.createSet uses a ScanBamParam internally, but as a user you do not > have access to it. > > Martin > > > For the first method, a "destination" output file name has to be > designated. I don't want to create a separate file, so it appears that the > second method is what I want, as it allows control of which records are > imported and doesn't output a separate file. > > I set up a FilterRules object like so: > > filt <- FilterRules(list(isChr = function(x) rname(x) == "^Chr")) > > > I also set up a "which" variable to contain the seqnames in my BSgenome: > > which <- seqnames(BSgenome) > > > However, I'm getting stuck trying to apply either of these to > ScanBamParam. Perhaps I want ScanBamWhat instead of ScanBamParam? Can > someone clue me in about the proper syntax to accomplish this simple > filtering operation? > > Thanks for any help, > --Kelly > > ________________________________________ > From: bioconductor-bounces@r-project.org [ > bioconductor-bounces@r-project.org] on behalf of Vining, Kelly [ > Kelly.Vining@oregonstate.edu] > Sent: Tuesday, April 01, 2014 8:12 AM > To: 'Lukas Chavez' > Cc: Steve Lianoglou; bioconductor@r-project.org > Subject: Re: [BioC] MEDIPS.createSet error > > 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]] > > _______________________________________________ > 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 > > _______________________________________________ > 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 > > > > -- > Computational Biology / Fred Hutchinson Cancer Research Center > 1100 Fairview Ave. N. > PO Box 19024 Seattle, WA 98109 > > Location: Arnold Building M1 B861 > Phone: (206) 667-2793 > > > > > > > [[alternative HTML version deleted]]