[BioC] applyPileups invalid times argument error

[Mark Dunning]

Hi all,

I am trying to get per-base counts over a series of ranges using the
pileupAsGRanges function. I'm only interesting in certain regions for
each bam file, so have written a script to extract the pileup from
each bam file for the regions of interest to that bam (the gr2 object
below, which changes according to bam file).

However, I'm having issues creating pileup from one of my ranges
objects. To my eye, it looks to be a valid GRanges object

> gr2
GRanges with 12 ranges and 4 metadata columns:
        seqnames                 ranges strand |                     Sample
           <Rle>              <IRanges>  <Rle> |                   <factor>
   F1_W    chr18 [ 48604746,  48604747]      * |            EN-438-11_Blood
   F2_W     chr2 [125320840, 125320841]      * |            EN-438-11_Blood
   F3_W    chr17 [  7577114,   7577115]      * | EN-454-12_EXP0628_TF_Blood
   F4_W     chr1 [ 27099124,  27099125]      * | EN-454-12_EXP0628_TF_Blood
   F5_W     chr9 [120474984, 120474985]      * | EN-454-12_EXP0628_TF_Blood
   F6_W     chr1 [248039414, 248039415]      * | EN-454-12_EXP0628_TF_Blood
   F7_W    chr19 [ 11130352,  11130353]      * |            EN-460-12_Blood
   F8_W    chr19 [ 11141514,  11141515]      * |            EN-460-12_Blood
   F9_W     chr8 [ 89058912,  89058913]      * |            EN-460-12_Blood
  F10_W    chr20 [ 23016541,  23016542]      * |  EN-64-10_EXP0628_TF_Blood
  F11_W    chr10 [118225601, 118225602]      * |  EN-64-10_EXP0628_TF_Blood
  F12_W    chr17 [  7578188,   7578189]      * |  EN-81-10_EXP0628_TF_Blood
            Barcode Ref_base Alt_base
        <character> <factor> <factor>
   F1_W      FLD283        G        T
   F2_W      FLD283        T        G
   F3_W      FLD283        C        T
   F4_W      FLD283        G        A
   F5_W      FLD283        C        A
   F6_W      FLD283        G        A
   F7_W      FLD283        T        C
   F8_W      FLD283        A        G
   F9_W      FLD283        C        T
  F10_W      FLD283        A        G
  F11_W      FLD283        A        T
  F12_W      FLD283        C        A


> pileupAsGRanges(bam=bams[85], region=gr2,maxDepth=.Machine$integer.max,minBaseQuality=30, minMapQuality=30)
Error: applyPileups: invalid 'times' argument
In addition: Warning messages:
1: In start(regions):end(regions) :
  numerical expression has 12 elements: only the first used
2: In start(regions):end(regions) :
  numerical expression has 12 elements: only the first used


However, the pileup seems to work for each element in the GRanges
object individually if I call them one-by-one without the error or the
warning.

e.g.

> pileupAsGRanges(bam=bams[85], region=gr2[1,],maxDepth=.Machine$integer.max,minBaseQuality=30, minMapQuality=30)
GRanges with 2 ranges and 7 metadata columns:
      seqnames             ranges strand |         A         C         G
         <Rle>          <IRanges>  <Rle> | <integer> <integer> <integer>
  [1]    chr17 [7577539, 7577539]      + |         0         0        27
  [2]    chr17 [7577540, 7577540]      + |         0         0        27
              T         N     depth
      <integer> <integer> <numeric>
  [1]         0         0        27
  [2]         0         0        27
                                                          bam
                                                  <character>
  [1] bamfiles/SLX-6363.FLD0187.1093.s_1.bwa.homo_sapiens.bam
  [2] bamfiles/SLX-6363.FLD0187.1093.s_1.bwa.homo_sapiens.bam
  ---
  seqlengths:
   chr17
      NA

I'm struggling to see why the pileup using the whole gr2 object as an
input isn't working.

Thanks a lot,

Mark

> sessionInfo()
R version 2.15.2 (2012-10-26)
Platform: x86_64-pc-linux-gnu (64-bit)

locale:
 [1] LC_CTYPE=en_GB.UTF-8       LC_NUMERIC=C
 [3] LC_TIME=en_GB.UTF-8        LC_COLLATE=en_GB.UTF-8
 [5] LC_MONETARY=en_GB.UTF-8    LC_MESSAGES=en_GB.UTF-8
 [7] LC_PAPER=C                 LC_NAME=C
 [9] LC_ADDRESS=C               LC_TELEPHONE=C
[11] LC_MEASUREMENT=en_GB.UTF-8 LC_IDENTIFICATION=C

attached base packages:
[1] stats     graphics  grDevices utils     datasets  methods   base

other attached packages:
[1] gdata_2.12.0         biovizBase_1.6.2     Rsamtools_1.10.2
[4] Biostrings_2.26.3    GenomicRanges_1.10.7 IRanges_1.16.6
[7] BiocGenerics_0.4.0

loaded via a namespace (and not attached):
 [1] AnnotationDbi_1.20.7   Biobase_2.18.0         biomaRt_2.14.0
 [4] bitops_1.0-5           BSgenome_1.26.1        cluster_1.14.2
 [7] colorspace_1.2-1       DBI_0.2-5              dichromat_2.0-0
[10] GenomicFeatures_1.10.2 grid_2.15.2            gtools_2.7.0
[13] Hmisc_3.10-1           labeling_0.1           lattice_0.20-10
[16] munsell_0.4            parallel_2.15.2        plyr_1.8
[19] RColorBrewer_1.0-5     RCurl_1.95-4.1         RSQLite_0.11.2
[22] rtracklayer_1.18.2     scales_0.2.3           stats4_2.15.2
[25] stringr_0.6.2          tools_2.15.2           XML_3.95-0.2
[28] zlibbioc_1.4.0