Hi,

I am using ChIPseeker to find the enrichment of genomic annotations in several Chip peak experiments. I would like to specify my own TxDB and I have used GenomicFeatures to create this (function: makeTxDbFromGRanges). Also, I have used readPeakFile to read all my peaks and save them in a list as the Vignette shows. 

Everything seems to work fine until I try to find enrichment of different genomic annotations within the peaks. I get the following error when I try the command copied below, so it seems as the txdb is not specified correctly (I am trying both the file path for the database and the txdb that I had created). I tried to specify the path both as a file where the sqlite is saved and after loading it using loadDb, but neither works. What am I doing wrong? txdb worked fine when I used it in annotatePeak, so I don't know why it is not working here...

Also, the Vignette shows the use of GRanges directly for other functions in this package (so for example peaks as a list of GRange object), can I also specify GRanges directly in this function too, instead of BED files for this function? 

Thank you.

     enrichPeakOverlap(queryPeak=genomicAnnotationsBed, targetPeak=pathBedPeaks,TxDB=txdb, pAdjustMethod = "BH", nShuffle =1000, chainFile= NULL, verbose = FALSE)

# Error using the self-specified txdb:

Error in enrichOverlap.peak.internalquery.gr, target.gr, TxDb, nShuffle,  : 

  unused argument (TxDB = <S4 object of class "TxDb">)

In addition: Warning message:

In loadTxDb(TxDb) :

  >> TxDb is not specified, use 'TxDb.Hsapiens.UCSC.hg19.knownGene' by default...

# Error when trying the sqlite path for the txdb:

Error in enrichOverlap.peak.internalquery.gr, target.gr, TxDb, nShuffle,  : 

  unused argument (TxDB = "txdb.sqlite")

In addition: Warning message:

In loadTxDb(TxDb) :

  >> TxDb is not specified, use 'TxDb.Hsapiens.UCSC.hg19.knownGene' by default...

> sessionInfo()  

R version 3.1.2 (2014-10-31)

Platform: x86_64-unknown-linux-gnu (64-bit)

locale:

[1] C

attached base packages:

[1] stats4    parallel  stats     graphics  grDevices utils     datasets 

[8] methods   base     

other attached packages:

 [1] XVector_0.6.0          ChIPseeker_1.5.11      GenomicFeatures_1.18.7

 [4] AnnotationDbi_1.28.2   Biobase_2.28.0         GenomicRanges_1.18.4  

 [7] GenomeInfoDb_1.2.5     IRanges_2.0.1          S4Vectors_0.4.0       

[10] BiocGenerics_0.12.1   

loaded via a namespace (and not attached):

 [1] BBmisc_1.9                             

 [2] BatchJobs_1.6                          

 [3] BiocParallel_1.0.3                     

 [4] Biostrings_2.34.1                      

 [5] DBI_0.3.1                              

 [6] GenomicAlignments_1.2.2                

 [7] KernSmooth_2.23-13                     

 [8] MASS_7.3-35                            

 [9] R6_2.1.1                               

[10] RColorBrewer_1.1-2                     

[11] RCurl_1.95-4.7                         

[12] RSQLite_1.0.0                          

[13] Rcpp_0.12.1                            

[14] Rsamtools_1.18.3                       

[15] TxDb.Hsapiens.UCSC.hg19.knownGene_3.0.0

[16] UpSetR_0.0.5                           

[17] XML_3.98-1.3                           

[18] assertthat_0.1                         

[19] base64enc_0.1-3                        

[20] biomaRt_2.24.1                         

[21] bitops_1.0-6                           

[22] boot_1.3-13                            

[23] brew_1.0-6                             

[24] caTools_1.17.1                         

[25] checkmate_1.6.2                        

[26] codetools_0.2-9                        

[27] colorspace_1.2-6                       

[28] digest_0.6.8                           

[29] dplyr_0.4.3                            

[30] fail_1.3                               

[31] foreach_1.4.2                          

[32] gdata_2.17.0                           

[33] ggplot2_1.0.1                          

[34] gplots_2.17.0                          

[35] grid_3.1.2                             

[36] gridBase_0.4-7                         

[37] gridExtra_2.0.0                        

[38] gtable_0.1.2                           

[39] gtools_3.5.0                           

[40] iterators_1.0.7                        

[41] magrittr_1.5                           

[42] munsell_0.4.2                          

[43] plotrix_3.5-12                         

[44] plyr_1.8.3                             

[45] proto_0.3-10                           

[46] reshape2_1.4.1                         

[47] rtracklayer_1.26.3                     

[48] scales_0.3.0                           

[49] sendmailR_1.2-1                        

[50] stringi_0.5-5                          

[51] stringr_1.0.0                          

[52] tools_3.1.2                            

[53] zlibbioc_1.12.0                       

Hi Francesca,

The error message is pretty clear:

  Error in enrichOverlap.peak.internalquery.gr, target.gr, TxDb, nShuffle,  : 

    unused argument (TxDB = <S4 object of class "TxDb">)

The enrichPeakOverlap() function has no TxDB argument. The correct spelling for this argument is TxDb.

@Guangchuang Yu: Do you really need the ellipsis at the end of your argument list? The man page for enrichPeakOverlap() only says "additional parameter" which leaves the user with no clue about what kind of additional parameter can be specified. Furthermore it seems to me that any additional parameter is actually ignored. Would you consider getting rid of the ellipsis? Then the user would get a more straightforward error in case s/he misspells an argument. Thanks!

H.

Thank you Herve', it is working now with the BED files and the TxDb argument in the correct spelling (I only get a warning copied below which I am not sure what it means...)

But I was wondering if I could specify GRanges directly instead of having to save the BED files or a TxDb object, or whether I need to change the function to be able to use the GRanges instead of BED files.

I am asking because the annotations I use for queryPeak are GRanges and the peaks list for targetPeak is a list of GRange objects as shown in the Vignette. To use this function, I need to save the annotations I am using in the targetPeak as BED files first, so I was just wondering whether there is a simpler way I am not seeing...

And for the TxDb, I am trying to modify the functions for example of "getPromoters" to be able to apply these directly to GRange objects since also my TxDb is in Grange...

Thank you!!

Warning message:

In mclapply(seq_along(idx), function(j) { :

  all scheduled cores encountered errors in user code

Thank you very much!

My reasoning for asking about getPromoters is because when I use this function after subsetting my GRange object to only include exons and converting to TxDb (using the function you suggest), and then apply getTagMatrix using the result of getPromoters, I get an error, is it because I need to split the GRange object first by something before creating the TxDb?

Thanks again very much for all the help, and for the brilliant package.

getTagMatrix(peak, windows=promoter)

Error in split.default(1:length(windows), as.factor(seqnames(windows))) : 

  group length is 0 but data length > 0

In addition: Warning message:

In .Seqinfo.mergexy(x, y) :

  The 2 combined objects have no sequence levels in common. (Use

  suppressWarnings() to suppress this warning.)

Yes sorry, I will try to explain better what I am trying to do with the code, I think my error comes from converting the incorrect TxDb from GenomicFeatures that needs to be used to getPromoter function...

    library("GenomicFeatures")

    # Download the version 19 gencode gtf file from gencode website (ftp://ftp.sanger.ac.uk/pub/gencode/Gencode_human/release_19/gencode.v19.annotation.gtf.gz)

    gencode = makeTxDbFromGFF("path/gtf/gencode.gtf")
    # Select exons
    exons.gencode = exons(gencode)

    # Add a "type" so that can make TxDb
    exons.gencode$type="exon_id"

    txdb=makeTxDbFromGRanges(exons.gencode)

    saveDb(gencode, file="exon.gencode_human_v19.sqlite")

    # Now use ChIPseeker to find promoters and overlap with peaks
    library(ChIPseeker)
    files <- getSampleFiles()
    peak <- readPeakFile(files[[4]])
    tagMatrix <- tagMatrixList[[4]]

    txdb = loadDb("exon.gencode_human_v19.sqlite")
   promoter <- getPromoters(TxDb=txdb, upstream=3000, downstream=3000)
   tagMatrix <- getTagMatrix(peak, windows=promoter)

Oh I see, sorry... it makes sense now. 

Thank you so much for your help and the quick replies!

I look forward to extensive use of this package!

centering all peaks to the start region of exon is now supported, see https://github.com/GuangchuangYu/ChIPseeker/issues/16