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GAGE pathway analysis (R/Bioconductor): summarizeOverlaps error
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14 months ago
user31888 • 60
United States

I am trying to use the GAGE pathway analysis tutorial with 3 samples of paired-end RNA-Seq reads (placed in a "tophat_all" directory) that I mapped with TopHat (.bam files have been indexed).

An error occurred at the read count step:

> library(TxDb.Hsapiens.UCSC.hg19.knownGene)

> exByGn <- exonsBy(TxDb.Hsapiens.UCSC.hg19.knownGene, "gene")

> library(GenomicAlignments)

> fls <- list.files("tophat_all/", pattern="bam$", full.names =T)

> bamfls <- BamFileList(fls)

> flag <- scanBamFlag(isSecondaryAlignment=FALSE, isProperPair=TRUE)

> param <- ScanBamParam(flag=flag)

> gnCnt <- summarizeOverlaps(exByGn, bamfls, mode="Union", ignore.strand=TRUE, single.end=FALSE, param=param)

Error in validObject(.Object) : 
  invalid class "SummarizedExperiment0" object: 'assays' nrow differs from 'mcols' nrow

When I tried the same with only one sample I get a different error:

> gnCnt <- summarizeOverlaps(exByGn, bamfls, mode="Union", ignore.strand=TRUE, single.end=FALSE, param=param)

Error in array(x, c(length(x), 1L), if (!is.null(names(x))) list(names(x),  : 
  'data' must be of a vector type, was 'NULL'

Could someone explain me the error?

Does the error come from my reads (potential unpaired reads, different length...)?

Thanks !

Note:

(1) I am using the last version of R, Biconductor and its packages. Could it come from the newer versions of the packages that would not fit the tutorial anymore?

> sessionInfo()
R version 3.2.2 (2015-08-14)
Platform: x86_64-pc-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] GenomicAlignments_1.6.1                
 [2] Rsamtools_1.22.0                       
 [3] Biostrings_2.38.0                      
 [4] XVector_0.10.0                         
 [5] SummarizedExperiment_1.0.1             
 [6] TxDb.Hsapiens.UCSC.hg19.knownGene_3.2.2
 [7] GenomicFeatures_1.22.4                 
 [8] AnnotationDbi_1.32.0                   
 [9] Biobase_2.30.0                         
[10] GenomicRanges_1.22.1                   
[11] GenomeInfoDb_1.6.1                     
[12] IRanges_2.4.1                          
[13] S4Vectors_0.8.2                        
[14] BiocGenerics_0.16.1                    

loaded via a namespace (and not attached):
 [1] zlibbioc_1.16.0      BiocParallel_1.4.0   tools_3.2.2         
 [4] DBI_0.3.1            lambda.r_1.1.7       futile.logger_1.4.1 
 [7] rtracklayer_1.30.1   futile.options_1.0.0 bitops_1.0-6        
[10] RCurl_1.95-4.7       biomaRt_2.26.0       RSQLite_1.0.0       
[13] XML_3.98-1.3

(2) And the traceback after the error occurred:

> traceback()
26: stop(msg, ": ", errors, domain = NA)
25: validObject(.Object)
24: initialize(value, ...)
23: initialize(value, ...)
22: new("SummarizedExperiment0", NAMES = names, elementMetadata = rowData, 
        colData = colData, assays = assays, metadata = as.list(metadata))
21: new_SummarizedExperiment0(from@assays, names(from@rowRanges), 
        mcols(from@rowRanges), from@colData, from@metadata)
20: asMethod(object)
19: as(object, superClass)
18: slot(x, "assays")
17: is(slot(x, "assays"), "Assays")
16: .valid.SummarizedExperiment0.assays_current(x)
15: valid.func(object)
14: validityMethod(as(object, superClass))
13: anyStrings(validityMethod(as(object, superClass)))
12: validObject(.Object)
11: initialize(value, ...)
10: initialize(value, ...)
9: new("RangedSummarizedExperiment", rowRanges = rowRanges, colData = colData, 
       assays = assays, elementMetadata = elementMetadata, metadata = as.list(metadata))
8: .new_RangedSummarizedExperiment(assays, rowRanges, colData, metadata)
7: .local(assays, ...)
6: SummarizedExperiment(assays = SimpleList(counts = counts), rowRanges = features, 
       colData = colData)
5: SummarizedExperiment(assays = SimpleList(counts = counts), rowRanges = features, 
       colData = colData)
4: .dispatchBamFiles(features, reads, mode, match.arg(algorithm), 
       ignore.strand, inter.feature = inter.feature, singleEnd = singleEnd, 
       fragments = fragments, param = param, preprocess.reads = preprocess.reads, 
       ...)
3: .local(features, reads, mode, algorithm, ignore.strand, ...)
2: summarizeOverlaps(exByGn, bamfls, mode = "Union", ignore.strand = TRUE, 
       single.end = FALSE, param = param)
1: summarizeOverlaps(exByGn, bamfls, mode = "Union", ignore.strand = TRUE, 
       single.end = FALSE, param = param)
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