Hi,

On Friday, February 1, 2013, Matteo Carrara wrote:

> Dear Alejandro,
>
> thank you for the quick reply. After you mentioned the coherence of
> the input, I dug more in it. It looks like the problem was quite
> trivial: I accidentally left the "paired" option of the counting
> script to the default, event though my datasets were all paired-end. I
> have run the script again with the correct parameters and the loading
> ends successfully.
>
> Your question about the samples is far from being unrelated, since it
> has impact on the next steps of the analysis.
> When I wrote the message I was just testing the function, so I limited
> the loading to a single dataset. The real design comprises two
> different conditions with two biological replicates, each of them with
> two technical replicates, for a total of four datasets per condition.


Keep in mind that you are usually recommended to simply add counts from
techinical replicates together, and not to treat them as separate
replicates -- so technically, you will have  two replicates per condition.

HTH,
-Steve


I decided to use DEXSeq exactly to assess the differential exon usage
>
> Thank you again.
>
> Best,
> --
> Matteo Carrara
> PhD Student in Complex Systems for Life Sciences
> Department of Biotechnology and Health Sciences
> MBC - Molecular Biotechnology Center
> via Nizza, 52 Torino
> ITALY
>
>
>
> On Tue, Jan 29, 2013 at 6:49 PM, Alejandro Reyes
> <alejandro.reyes@embl.de> wrote:
> >
> > Dear Matteo Carrara,
> >
> > Could you add to this e-mail the first 10 and last 10 lines of your
> input files (both counts and annotation files produced by the python
> scripts)?
> >
> > On unrelated topics, I noticed that you have only one sample, what
> exactly do you want to do with DEXSeq in this case?
> > Note that DEXSeq is designed to test for differences in exon usage
> between different conditions with replicates.
> >
> > Best wishes,
> > Alejandro Reyes
> >
> >
> >> Hello,
> >>
> >> I have been trying to learn how to perform a differential expression
> >> analysis of RNA-seq data using the DEXSeq package lately and I
> encountered
> >> an unexpected behaviour in the function read.HTSeqCount: the function
> fails
> >> to load the file obtained from the python script "dexseq_count.py" with
> the
> >> following error message:
> >>
> >> Error in strsplit(rownames(dcounts), ":") : non-character argument
> >>
> >> I would really appreciate any pointers that might help me correct my
> code
> >> or my input files.
> >>
> >> Here is what I have done:
> >> - downloaded the mm9 GTF gene set from www.ensembl.org and run the
> script
> >> "dexseq_prepare_annotation.py"
> >> - mapped my raw RNA-seq reads on the mm9 genome using tophat, converting
> >> the output in sorted SAM format
> >> - run the script "dexseq_count.py" using the "flattened" GTF and the SAM
> >> file obtained before
> >> - loaded the dataset in R using the function read.HTSeqCount() as
> following:
> >>
> >> --------------------------------------
> >>>
> >>> library(DEXSeq)
> >>> wt<-read.HTSeqCount("./wt_mapped.counts", "WT",
> >>
> >> flattenedfile="./flattened_mm9.gtf")
> >>
> >>
> >> Error in strsplit(rownames(dcounts), ":") : non-character argument
> >> --------------------------------------
> >>
> >> As far as I could understand, the "pasilla" package, used for the
> examples
> >> in the vignette, provided a counts file under the name
> >> "pasilla_gene_counts.tsv". Loading that file, however, results in the
> same
> >> error message.
> >>
> >> All I could do was pinpointing the source of the error in the code of
> the
> >> function, although that did not help me in finding a solution or a
> >> workaround:
> >> After creating the data frame "dcounts" storing the counts and setting
> the
> >> row names, that same data frame is sub-set
> >>
> >>      dcounts <- dcounts[substr(rownames(dcounts), 1, 1) != "_",
> >>          ]
> >>
> >> This code, however changes the object dcounts in such a way that the
> >> "rownames()" function returns NULL. The next statement is then bound to
> >> fail since it requires rownames(dcounts) to be a character or a vector
> of
> >> characters:
> >>
> >>      genesrle <- sapply(strsplit(rownames(dcounts), ":"), "[[",
> >>          1)
> >>
> >> I am running R 2.15.2  and DEXSeq  1.4.0 from Bioconductor version 2.11,
> >> but I was able to reproduce this on the devel version of R (2013-01-22
> >> r61734) using DEXSeq_1.5.6 from Bioconductor version 2.12.
> >>
> >> ---------------------------------
> >>>
> >>> sessionInfo()
> >>
> >> R version 2.15.2 (2012-10-26)
> >> 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] stats     graphics  grDevices--
> Matteo Carrara
>
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>


-- 
Steve Lianoglou
Graduate Student: Computational Systems Biology
 | Memorial Sloan-Kettering Cancer Center
 | Weill Medical College of Cornell University
Contact Info: http://cbio.mskcc.org/~lianos/contact

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