Dear Surjyendu Ray,
The issue is corrected in package version 1.2.5 (stable) and version
1.3.13 (devel).
These will be available in a couple of days from Bioconductor. In the
meanwhile, the following code resolves the issue and gives the
expected results.
## libs
library(easyRNASeq)
library(RnaSeqTutorial)
library(BSgenome.Dmelanogaster.UCSC.dm3)
## conditions
conditions <- rep(c("A","B"),each=2)
names(conditions) <- dir(system.file("extdata",package="RnaSeqTutorial
"),pattern="[A,C,T,G]{6}\\.bam$")
## count
obj <- easyRNASeq(
filesDirectory=
system.file(
"extdata",package="RnaSeqTutorial"),
pattern="[A,C,T,G]{6}\\.bam$",
readLength=30L,
organism="Dmelanogaster",
chr.sizes=as.list(seqlengths(Dmelanogaster)),
annotationMethod="rda",
annotationFile=system.file("data","gAnnot.rda",packa
ge="RnaSeqTutorial"),
count="exons",outputFormat="edgeR",
format="bam",
normalize=FALSE,
conditions=conditions)
## calculate the normalization factors
obj <- calcNormFactors(obj)
## plot the normalization factor per sample pairs
apply(combn(rownames(obj$samples),2),2,function(co,obj){plotNormalizat
ionFactors(obj,co[1],co[2])},obj)
## plot the MDS
plotMDS.DGEList(obj, main = "MDS of all conditions", labels =
rownames(obj$samples))
## calculate the dispersion
obj <- estimateCommonDisp(obj)
## plot the dispersion estimate
obj <- estimateTagwiseDisp(obj)
plotDispersionEstimates(obj)
plotMeanVar(obj, show.raw.vars = TRUE, show.tagwise.vars = TRUE,
NBline = TRUE, main="Mean-variance (tag variances against
tag abundance)")
legend("bottomright",col=c("gray60","lightskyblue","darkred","dodgerbl
ue3",1),
pch=c("o","o","x",rep(NA,2)),lty=c(rep(NA,3),1,1),lwd=c(rep(NA,
3),4,1),
,pt.cex=0.6,c("raw tagwise variances","gene estimated
variance",
"100 bin averaged raw variance","common dispersion est.
var.",
"poisson variance"))
Best,
---------------------------------------------------------------
Nicolas Delhomme
Genome Biology Computational Support
European Molecular Biology Laboratory
Tel: +49 6221 387 8310
Email: nicolas.delhomme at embl.de
Meyerhofstrasse 1 - Postfach 10.2209
69102 Heidelberg, Germany
---------------------------------------------------------------
On 22 Aug 2012, at 23:17, Surjyendu Ray wrote:
> Dear Sir,
> Please do let me know what you think is going wrong in
the analysis.
> Thanking you,
> Yours faithfully,
> Surjyendu Ray.
>
> On Thu, Aug 16, 2012 at 5:13 PM, Surjyendu Ray <surjray at="" gmail.com=""> wrote:
> Dear Sir,
> The following is the output of the sessioninfo()
command:
>
> R version 2.15.1 (2012-06-22)
> Platform: x86_64-unknown-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] easyRNASeq_1.2.4 ShortRead_1.14.4
latticeExtra_0.6-19
> [4] RColorBrewer_1.0-5 lattice_0.20-6 Rsamtools_1.8.6
> [7] DESeq_1.8.3 locfit_1.5-8 BSgenome_1.24.0
> [10] GenomicRanges_1.8.12 Biostrings_2.24.1 IRanges_1.14.4
> [13] edgeR_2.6.10 limma_3.12.1 biomaRt_2.12.0
> [16] Biobase_2.16.0 genomeIntervals_1.12.0
BiocGenerics_0.2.0
> [19] intervals_0.13.3
>
> loaded via a namespace (and not attached):
> [1] annotate_1.34.1 AnnotationDbi_1.18.1 bitops_1.0-4.1
> [4] DBI_0.2-5 genefilter_1.38.0 geneplotter_1.34.0
> [7] grid_2.15.1 hwriter_1.3 RCurl_1.91-1
> [10] RSQLite_0.11.1 splines_2.15.1 stats4_2.15.1
> [13] survival_2.36-14 XML_3.9-4 xtable_1.7-0
> [16] zlibbioc_1.2.0
>
> Please advise.
>
> Thanking you,
> Yours faithfully,
> Surjyendu Ray.
>
> On Thu, Aug 16, 2012 at 3:48 AM, Nicolas Delhomme <delhomme at="" embl.de=""> wrote:
> Dear Surjyendu Ray,
>
> It seems to be an issue with the edgeR API. I would need more
information from you, can you please run in your R session:
>
> library(easyRNASeq)
> sessionInfo()
>
> and send me the results?
>
> Thanks,
>
> Nico
>
> P.S. I've Cc'ed the Bioconductor mailing list as it might help other
users. If you do not know this mailing list and are doing analyses in
R/Bioc, I would advise you to subscribe to it.
>
> ---------------------------------------------------------------
> Nicolas Delhomme
>
> Genome Biology Computational Support
>
> European Molecular Biology Laboratory
>
> Tel: +49 6221 387 8310
> Email: nicolas.delhomme at embl.de
> Meyerhofstrasse 1 - Postfach 10.2209
> 69102 Heidelberg, Germany
> ---------------------------------------------------------------
>
>
>
>
>
> On Aug 16, 2012, at 5:53 AM, Surjyendu Ray wrote:
>
> > Dear Sir,
> > I was running an experiment with two samples and
three replicates for each sample. Here are the commands I used:
> >
> > conditions = c( "HCV1", "HCV1", "HCV1", "HCM1", "HCM1", "HCM1" )
> > names(conditions) <- c( "accepted_hits_ATCACG.sorted.bam",
"accepted_hits_CGATGT.sorted.bam", "accepted_hits_TTAGGC.sorted.bam",
"accepted_hits_CAGATC.sorted.bam", "accepted_hits_ACTTGA.sorted.bam",
"accepted_hits_GATCAG.sorted.bam" )
> >
> > and the easyRNASeq command:
> > FM_HCV1_HCM1.count.table <- easyRNASeq( filesDirectory =
"/panfs/storage.local/genacc/home/sr09m/Biomedicine research/New
Drosophila analysis/Tophat_temp", organism = "Dmelanogaster",
chr.sizes = as.list( seqlengths( Dmelanogaster )), readLength = 76L,
annotationMethod = "gff", annotationFile =
"/panfs/storage.local/genacc/home/sr09m/Biomedicine research/New
Drosophila analysis/dmel-all-r5.46.gff", format = "bam", count =
"exons", filenames = c( "accepted_hits_ATCACG.sorted.bam",
"accepted_hits_CGATGT.sorted.bam", "accepted_hits_TTAGGC.sorted.bam",
"accepted_hits_CAGATC.sorted.bam", "accepted_hits_ACTTGA.sorted.bam",
"accepted_hits_GATCAG.sorted.bam" ), normalize = TRUE, outputFormat =
"edgeR", conditions = conditions )
> >
> > However, I got an error:
> > Error in match.arg(trend, c("none", "movingave", "tricube")) :
> > 'arg' must be NULL or a character vector
> >
> > The entire log of outputs is:
> > Checking arguments...
> > Fetching annotations...
> > Read 12568191 records
> > Summarizing counts...
> > Processing accepted_hits_ATCACG.sorted.bam
> > Processing accepted_hits_CGATGT.sorted.bam
> > Processing accepted_hits_TTAGGC.sorted.bam
> > Processing accepted_hits_CAGATC.sorted.bam
> > Processing accepted_hits_ACTTGA.sorted.bam
> > Processing accepted_hits_GATCAG.sorted.bam
> > Preparing output
> > Calculating library sizes from column totals.
> > Normalizing counts
> > Error in match.arg(trend, c("none", "movingave", "tricube")) :
> > 'arg' must be NULL or a character vector
> >
> > Please advise as to what may be going wrong.
> > Thanking you,
> > Yours faithfully,
> > Surjyendu Ray.
>
>
>
