GAGE pathway analysis (R/Bioconductor): summarizeOverlaps error

[user 31888]

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 !

Notes:
(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) Here is 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)

[gringer]

This error suggests that one of your two input arguments is empty. Try the following two commands to see if anything looks odd:

> str(exByGn)
> str(bamfls)

[user 31888]

Thanks ginger for your reply !

Here are the outputs:

> str(exByGn)
Formal class 'GRangesList' [package "GenomicRanges"] with 5 slots
  ..@ unlistData     :Formal class 'GRanges' [package "GenomicRanges"] with 6 slots
  .. .. ..@ seqnames       :Formal class 'Rle' [package "S4Vectors"] with 4 slots
  .. .. .. .. ..@ values         : Factor w/ 93 levels "chr1","chr2",..: 19 8 20 18 1 23 3 14 15 11 ...
  .. .. .. .. ..@ lengths        : int [1:19380] 15 2 12 17 17 4 1 11 10 21 ...
  .. .. .. .. ..@ elementMetadata: NULL
  .. .. .. .. ..@ metadata       : list()
  .. .. ..@ ranges         :Formal class 'IRanges' [package "IRanges"] with 6 slots
  .. .. .. .. ..@ start          : int [1:272776] 58858172 58858719 58859832 58860934 58861736 58862757 58863649 58864294 58864658 58864770 ...
  .. .. .. .. ..@ width          : int [1:272776] 224 288 663 1084 282 297 273 270 36 96 ...
  .. .. .. .. ..@ NAMES          : NULL
  .. .. .. .. ..@ elementType    : chr "integer"
  .. .. .. .. ..@ elementMetadata: NULL
  .. .. .. .. ..@ metadata       : list()
  .. .. ..@ strand         :Formal class 'Rle' [package "S4Vectors"] with 4 slots
  .. .. .. .. ..@ values         : Factor w/ 3 levels "+","-","*": 2 1 2 1 2 1 2 1 2 1 ...
  .. .. .. .. ..@ lengths        : int [1:11717] 15 2 46 5 11 87 1 4 1 8 ...
  .. .. .. .. ..@ elementMetadata: NULL
  .. .. .. .. ..@ metadata       : list()
  .. .. ..@ elementMetadata:Formal class 'DataFrame' [package "S4Vectors"] with 6 slots
  .. .. .. .. ..@ rownames       : NULL
  .. .. .. .. ..@ nrows          : int 272776
  .. .. .. .. ..@ listData       :List of 2
  .. .. .. .. .. ..$ exon_id  : int [1:272776] 250809 250810 250811 250812 250813 250814 250815 250816 250817 250818 ...
  .. .. .. .. .. ..$ exon_name: chr [1:272776] NA NA NA NA ...
  .. .. .. .. ..@ elementType    : chr "ANY"
  .. .. .. .. ..@ elementMetadata: NULL
  .. .. .. .. ..@ metadata       : list()
  .. .. ..@ seqinfo        :Formal class 'Seqinfo' [package "GenomeInfoDb"] with 4 slots
  .. .. .. .. ..@ seqnames   : chr [1:93] "chr1" "chr2" "chr3" "chr4" ...
  .. .. .. .. ..@ seqlengths : int [1:93] 249250621 243199373 198022430 191154276 180915260 171115067 159138663 146364022 141213431 135534747 ...
  .. .. .. .. ..@ is_circular: logi [1:93] NA NA NA NA NA NA ...
  .. .. .. .. ..@ genome     : chr [1:93] "hg19" "hg19" "hg19" "hg19" ...
  .. .. ..@ metadata       : list()
  ..@ elementMetadata:Formal class 'DataFrame' [package "S4Vectors"] with 6 slots
  .. .. ..@ rownames       : NULL
  .. .. ..@ nrows          : int 23459
  .. .. ..@ listData       : Named list()
  .. .. ..@ elementType    : chr "ANY"
  .. .. ..@ elementMetadata: NULL
  .. .. ..@ metadata       : list()
  ..@ partitioning   :Formal class 'PartitioningByEnd' [package "IRanges"] with 5 slots
  .. .. ..@ end            : int [1:23459] 15 17 29 46 63 67 68 79 89 110 ...
  .. .. ..@ NAMES          : chr [1:23459] "1" "10" "100" "1000" ...
  .. .. ..@ elementType    : chr "integer"
  .. .. ..@ elementMetadata: NULL
  .. .. ..@ metadata       : list()
  ..@ elementType    : chr "GRanges"
  ..@ metadata       :List of 1
  .. ..$ genomeInfo:List of 19
  .. .. ..$ Db type                                 : chr "TxDb"
  .. .. ..$ Supporting package                      : chr "GenomicFeatures"
  .. .. ..$ Data source                             : chr "UCSC"
  .. .. ..$ Genome                                  : chr "hg19"
  .. .. ..$ Organism                                : chr "Homo sapiens"
  .. .. ..$ Taxonomy ID                             : chr "9606"
  .. .. ..$ UCSC Table                              : chr "knownGene"
  .. .. ..$ Resource URL                            : chr "http://genome.ucsc.edu/"
  .. .. ..$ Type of Gene ID                         : chr "Entrez Gene ID"
  .. .. ..$ Full dataset                            : chr "yes"
  .. .. ..$ miRBase build ID                        : chr "GRCh37"
  .. .. ..$ transcript_nrow                         : chr "82960"
  .. .. ..$ exon_nrow                               : chr "289969"
  .. .. ..$ cds_nrow                                : chr "237533"
  .. .. ..$ Db created by                           : chr "GenomicFeatures package from Bioconductor"
  .. .. ..$ Creation time                           : chr "2015-10-07 18:11:28 +0000 (Wed, 07 Oct 2015)"
  .. .. ..$ GenomicFeatures version at creation time: chr "1.21.30"
  .. .. ..$ RSQLite version at creation time        : chr "1.0.0"
  .. .. ..$ DBSCHEMAVERSION                         : chr "1.1"
> str(bamfls)
Formal class 'BamFileList' [package "Rsamtools"] with 4 slots
  ..@ listData       :List of 3
  .. ..$ sample_1.bam :Reference class 'BamFile' [package "Rsamtools"] with 8 fields
  .. .. ..$ .extptr         :<externalptr> 
  .. .. ..$ path            : chr "tophat_all//sample_1.bam"
  .. .. ..$ index           : chr "tophat_all//sample_1.bam.bai"
  .. .. ..$ yieldSize       : int NA
  .. .. ..$ obeyQname       : logi FALSE
  .. .. ..$ asMates         : logi FALSE
  .. .. ..$ qnamePrefixEnd  : chr NA
  .. .. ..$ qnameSuffixStart: chr NA
  .. .. ..and 14 methods.
  .. ..$ sample_2.bam    :Reference class 'BamFile' [package "Rsamtools"] with 8 fields
  .. .. ..$ .extptr         :<externalptr> 
  .. .. ..$ path            : chr "tophat_all//sample_2.bam"
  .. .. ..$ index           : chr "tophat_all//sample_2.bam.bai"
  .. .. ..$ yieldSize       : int NA
  .. .. ..$ obeyQname       : logi FALSE
  .. .. ..$ asMates         : logi FALSE
  .. .. ..$ qnamePrefixEnd  : chr NA
  .. .. ..$ qnameSuffixStart: chr NA
  .. .. ..and 14 methods.
  .. ..$ sample_3.bam:Reference class 'BamFile' [package "Rsamtools"] with 8 fields
  .. .. ..$ .extptr         :<externalptr> 
  .. .. ..$ path            : chr "tophat_all//sample_3.bam"
  .. .. ..$ index           : chr "tophat_all//sample_3.bam.bai"
  .. .. ..$ yieldSize       : int NA
  .. .. ..$ obeyQname       : logi FALSE
  .. .. ..$ asMates         : logi FALSE
  .. .. ..$ qnamePrefixEnd  : chr NA
  .. .. ..$ qnameSuffixStart: chr NA
  .. .. ..and 14 methods.
  ..@ elementType    : chr "BamFile"
  ..@ elementMetadata: NULL
  ..@ metadata       : list()

[gringer]

There's something odd about sample_2.bam:

  .. .. ..$ path            : chr "GAGE//sample_2.bam"
  .. .. ..$ index           : chr "GAGE//sample_2.bai"

I think this should be:

  .. .. ..$ path            : chr "GAGE//sample_2.bam"
  .. .. ..$ index           : chr "GAGE//sample_2.bam.bai"

If that's not the problem, I can't see anything else obviously out of place that might indicate an empty list.

[user 31888]

My mistake, sorry.

It is actually 'sample_2.bam.bai' (I edited my previous post)

[user 31888]

Stupid thought: could it be something like EOF or other formatting issue that differs between my sample.bam files and TxDb.Hsapiens.UCSC.hg19.knownGene?

(I am using a Linux cluster)

[Enmity_LD]

Hi! user 31888, Does your issue being solved? if it does ,colud you please provide me some advice? I meet the same question ,Thank you

[user 31888]

maybe see:
https://support.bioconductor.org/p/74677/#75002

But I ended up doing a fresh install of pretty much everything (R, Python, Bioconductor and associated packages), and it worked.