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  • easyRNASeq errors / experiences

    Hi all,

    At the moment I'm trying to set up easyRNASeq + edgeR to analyse my paired-end data using R. I'm following the easyRNASeq manual to acquire a table of read counts which can then be used as DGElist object for edgeR.

    Unfortunately, I do not even get close to the point of obtaining the DGElist object, as the easyRNASeq function crashes with the following error:

    Code:
    Checking arguments... 
    Fetching annotations... 
    Computing gene models... 
    Summarizing counts... 
    Processing EMC_18_alignment.bam 
    Updating the read length information. 
    The alignments are gapped. 
    Minimum length of 1 bp. 
    Maximum length of 101 bp. 
    Error in mk_singleBracketReplacementValue(x, value) : 
      'value' must be a CompressedIntegerList object
    In addition: Warning messages:
    1: In easyRNASeq(organism = "Hsapiens", annotationMethod = "biomaRt",  :
      There are 16696 synthetic exons as determined from your annotation that overlap! This implies that some reads will be counted more than once! Is that really what you want?
    2: In fetchCoverage(rnaSeq, format = format, filename = filename, filter = filter,  :
      You enforce UCSC chromosome conventions, however the provided alignments are not compliant. Correcting it.
    Did anyone experience a similar error message yet?
    My bamfiles list consists of 4 samples aligned via GSNAP and here's how I run the function itself:

    Code:
    count.genes <- easyRNASeq(organism="Hsapiens",
                         annotationMethod="biomaRt",
                         gapped=TRUE, count="genes",
                         summarization="geneModels",
                         filesDirectory=getwd(),
                         filenames=bamfiles,
                         outputFormat="RNAseq")
    I use the devel version of easyRNASeq since it supports varying read lengths.


    Any help is greatly appreciated.


    Code:
    > sessionInfo()
    R version 2.15.1 (2012-06-22)
    Platform: x86_64-pc-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=C                 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] BiocInstaller_1.5.12               BSgenome.Hsapiens.UCSC.hg19_1.3.19
     [3] easyRNASeq_1.3.14                  ShortRead_1.15.11                 
     [5] latticeExtra_0.6-24                RColorBrewer_1.0-5                
     [7] Rsamtools_1.9.30                   DESeq_1.9.14                      
     [9] lattice_0.20-10                    locfit_1.5-8                      
    [11] BSgenome_1.25.8                    GenomicRanges_1.9.65              
    [13] Biostrings_2.25.12                 IRanges_1.15.44                   
    [15] edgeR_2.99.8                       limma_3.13.20                     
    [17] biomaRt_2.13.2                     Biobase_2.17.7                    
    [19] genomeIntervals_1.13.3             BiocGenerics_0.3.1                
    [21] intervals_0.13.3                  
    
    loaded via a namespace (and not attached):
     [1] annotate_1.35.3       AnnotationDbi_1.19.37 bitops_1.0-4.1       
     [4] DBI_0.2-5             genefilter_1.39.0     geneplotter_1.35.1   
     [7] grid_2.15.1           hwriter_1.3           RCurl_1.91-1         
    [10] RSQLite_0.11.2        splines_2.15.1        stats4_2.15.1        
    [13] survival_2.36-14      tools_2.15.1          XML_3.9-4            
    [16] xtable_1.7-0          zlibbioc_1.3.0

  • #2
    I have got the same error:

    #get annotation
    RNASeq<- easyRNASeq(filesDirectory=getwd(),
    organism="Hsapiens",
    #chr.sizes=chr.sizes,
    #readLength=80L,
    annotationMethod="biomaRt",
    format="bam",
    count="genes",
    summarization="geneModels",
    filenames=bamfiles[1],
    outputFormat="RNAseq"
    )
    gAnnot <- genomicAnnotation(rnaSeq)




    Checking arguments...
    Fetching annotations...
    Computing gene models...
    Summarizing counts...
    Processing RU_009_final.sorted.bam
    Updating the read length information.
    The reads have been trimmed.
    Minimum length of 50 bp.
    Maximum length of 80 bp.
    Error in mk_singleBracketReplacementValue(x, value) :
    'value' must be a CompressedIntegerList object
    In addition: Warning messages:
    1: In easyRNASeq(filesDirectory = getwd(), organism = "Hsapiens", annotationMethod = "biomaRt", :
    You enforce UCSC chromosome conventions, however the provided chromosome size list is not compliant. Correcting it.
    2: In easyRNASeq(filesDirectory = getwd(), organism = "Hsapiens", annotationMethod = "biomaRt", :
    There are 16696 synthetic exons as determined from your annotation that overlap! This implies that some reads will be counted more than once! Is that really what you want?
    3: In fetchCoverage(rnaSeq, format = format, filename = filename, filter = filter, :
    You enforce UCSC chromosome conventions, however the provided alignments are not compliant. Correcting it.
    Last edited by hollandorange; 09-20-2012, 01:28 AM.

    Comment


    • #3
      Hi hollandorange,

      could you include which aligner (+ version) you used? I forgot to mention that I aligned to Hg19 with GSNAP (version 2012-07-12).

      Cheers

      Comment


      • #4
        Hi rboettcher,

        Thanks for your email pointing me to that thread.

        There indeed seem to be a bug in a sub-setting step when getting the reads' information.

        As I'm usually not scanning the seqanswers forum for posts related to easyRNASeq, a better place to post about it is the bioconductor mailing list (I've forwarded your post there). Let's go on with this discussion over there.

        Cheers,

        Nico

        Comment


        • #5
          Hi Rboettcher,

          The bam files that I used for easyRNAseq was generated from Tophat. I also wanted to use GSNAP, since I heard it is more accurate.

          Could you also forward me to the bioconductor email thread for this issue? thanks!

          Hollandorange

          Comment


          • #6
            Hi hollandorange,

            You can register for that mailing list there: http://www.bioconductor.org/help/mailing-list/ (the best option IMO) or follow it on GMANE there:



            Cheers,

            Nico

            Comment

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