summarizeOverlaps error

[Parharn]

Hi,
I am trying to test the "RNA-Seq Data Pathway and Gene-set Analysis Workflows" manual by Weijun Luo. I get an error as follow:

> gnCnt <- summarizeOverlaps(exByGn, bamfls, mode="Union", ignore.strand=TRUE, single.end=TRUE, param=param)
Error: could not find function "summarizeOverlaps"

Does anybody have any suggestions?

Thanks,
Parham

[dpryan]

You need to load GenomicRanges:

library(GenomicRanges)

[Parharn]

I did! Still it cannot find the function!
> library(GenomicRanges)
> gnCnt <- summarizeOverlaps(exByGn, bamfls, mode="Union", ignore.strand=TRUE, single.end=FALSE, param=param)
Error: could not find function "summarizeOverlaps"

Do you have any other suggestion?

[dpryan]

That's a first. Try starting a new R session.

[dpryan]

And if that doesn't work, post the output of sessionInfo()

[Parharn]

Alright, it didn't work yet. Here is the code that I am trying (I couldn't figure out how you put a code in box as your first reply) however here it is:

library(TxDb.Hsapiens.UCSC.hg19.knownGene)
exByGn <- exonsBy(TxDb.Hsapiens.UCSC.hg19.knownGene, "gene")
library(Rsamtools)
fls <- list.files("tophat_all/", pattern="bam$", full.names =T)
bamfls <- BamFileList(fls)
flag <- scanBamFlag(isNotPrimaryRead=FALSE, isProperPair=TRUE)
param <- ScanBamParam(flag=flag)
gnCnt <- summarizeOverlaps(exByGn, bamfls, mode="Union",
ignore.strand=TRUE, single.end=FALSE, param=param)
hnrnp.cnts=assay(gnCnt)

One thing I need to know is if I can load several libraries in the beginning of R?
The next thing is for loading Rsamtools it said two packages are required (XVector and Biostrings) which I had installed them previously but it asked my again, and I had to install them again. I don't know what's wrong, but just wanted to provide that info to you.

I am not developer, just trying to use linux and these softwares.

Thanks!

[dpryan]

Yes, you can load as many libraries as you want at the beginning. What's the output of sessionInfo()?

[Parharn]

All right, good to know!
Here it is:

sessionInfo()
R version 3.1.0 (2014-04-10)
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=en_GB.UTF-8 LC_NAME=C
[9] LC_ADDRESS=C LC_TELEPHONE=C
[11] LC_MEASUREMENT=en_GB.UTF-8 LC_IDENTIFICATION=C

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

other attached packages:
[1] Rsamtools_1.16.0
[2] Biostrings_2.32.0
[3] XVector_0.4.0
[4] TxDb.Hsapiens.UCSC.hg19.knownGene_2.14.0
[5] GenomicFeatures_1.16.2
[6] AnnotationDbi_1.26.0
[7] Biobase_2.24.0
[8] GenomicRanges_1.16.3
[9] GenomeInfoDb_1.0.2
[10] IRanges_1.22.8
[11] BiocGenerics_0.10.0

loaded via a namespace (and not attached):
[1] BatchJobs_1.2 BBmisc_1.6 BiocParallel_0.6.1
[4] biomaRt_2.20.0 bitops_1.0-6 brew_1.0-6
[7] BSgenome_1.32.0 codetools_0.2-8 DBI_0.2-7
[10] digest_0.6.4 fail_1.2 foreach_1.4.2
[13] GenomicAlignments_1.0.1 iterators_1.0.7 plyr_1.8.1
[16] Rcpp_0.11.1 RCurl_1.95-4.1 RSQLite_0.11.4
[19] rtracklayer_1.24.2 sendmailR_1.1-2 stats4_3.1.0
[22] stringr_0.6.2 tools_3.1.0 XML_3.98-1.1
[25] zlibbioc_1.10.0
>

[Michael Love]

summarizeOverlaps() moved to the GenomicAlignments package for Bioc 2.14 / R 3.1

library(GenomicAlignments)

http://www.bioconductor.org/packages/devel/bioc/html/GenomicAlignments.html

[Parharn]

Thanks Michael, it worked. But then I got another error regarding the BAM file:

gnCnt <- summarizeOverlaps(exByGn, bamfls, mode="Union", ignore.strand=TRUE, single.end=FALSE, param=param)
Error: 1 errors; first error:
Error in value[[3L]](cond): failed to open BamFile: file(s) do not exist:
'tophat_all//ERR127302.bam'

For more information, use bplasterror(). To resume calculation, re-call
the function and set the argument 'BPRESUME' to TRUE or wrap the
previous call in bpresume().

First traceback:
34: summarizeOverlaps(exByGn, bamfls, mode = "Union", ignore.strand = TRUE,
single.end = FALSE, param = param)
33: summarizeOverlaps(exByGn, bamfls, mode = "Union", ignore.strand = TRUE,
single.end = FALSE, param = param)
32: .local(features, reads, mode, ignore.strand, ...)
31: .summarizeOverlaps_BamFileList(features, reads, mode, ignore.strand = ignore.strand,
inter.feature = inter.feature, singleEnd = singleEnd, fragments = fragments,
param = param, ...)
30: .dispatchBamFiles(features, reads, mode, ignore.strand, inter.feature = inter.feature,
singleEnd = singleEnd, fragments = fragments, param = pa

Do you have any advice for it?

[Michael Love]

Well, you'll have to make sure the files in bamfls (e.g. 'tophat_all/ERR127302.bam') exist and are correctly specified relative to your current working directory in R:

getwd()

[Parharn]

Great, I fixed that. Thanks a lot!