Dear All,
I have RNA-seq data for 20 bumblebee samples divided into treatment and control. I am interested in differential exon usage. Unfortunately the bumblebee genome is not at the stage where I can do a complete DEXSeq analysis genome wide. I decided to look at a single gene where I have a decent gene model. Using TopHat2 and the python scripts included in DEXSeq I was able to produce an ExonCountSet object.

>head(counts(ecs))
                 K61 K62 K63 K83 Q61 Q62 Q63 Q81 Q82
XLOC_000001:E001   1   0   1   0   1   0   0   0   0
XLOC_000001:E002   1   0   0   0   2   1   0   1   1
XLOC_000001:E003   0   0   0   0   3   0   0   0   0
XLOC_000001:E004   1   0   1   0   1   0   0   1   0
XLOC_000001:E005   1   3   3   2   3   0   0   0   0
XLOC_000001:E006   0   0   0   0   0   0   0   0   0

As I expected, its all come crashing down round my ears at the analysis stage

> sizeFactors(ecs)
K61 K62 K63 K83 Q61 Q62 Q63 Q81 Q82
 NA  NA  NA  NA  NA  NA  NA  NA  NA

I tried using the shorth value

> ecs<-estimateSizeFactors(ecs,(locfunc=genefilter::shorth((counts(ecs)),tie.action="min")))
Error in .local(object, ...) : unused argument (0.2)

I understand that my main problem is that the DEXSeq analysis should be genome wide (my data has too few counts).

Is there a way to use DEXSeq to ONLY look at a single gene? If not can anyone think of a statistical way to analyse my data (cross tabs? How should I normalise?)

Thanks for your time

Eamonn


> sessionInfo()
R version 3.0.0 (2013-04-03)
Platform: x86_64-apple-darwin10.8.0 (64-bit)

locale:
[1] en_GB.UTF-8/en_GB.UTF-8/en_GB.UTF-8/C/en_GB.UTF-8/en_GB.UTF-8

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

other attached packages:
[1] genefilter_1.42.0  DEXSeq_1.6.0       Biobase_2.20.0     BiocGenerics_0.6.0

loaded via a namespace (and not attached):
 [1] annotate_1.38.0      AnnotationDbi_1.22.5 biomaRt_2.16.0       Biostrings_2.28.0    bitops_1.0-5         DBI_0.2-7
 [7] GenomicRanges_1.12.3 hwriter_1.3          IRanges_1.18.1       RCurl_1.95-4.1       Rsamtools_1.12.3     RSQLite_0.11.3
[13] splines_3.0.0        statmod_1.4.17       stats4_3.0.0         stringr_0.6.2        survival_2.37-4      tools_3.0.0
[19] XML_3.95-0.2         xtable_1.7-1         zlibbioc_1.6.0
Dr Eamonn Mallon
Lecturer in Evolutionary Biology
Adrian 220
Biology Department
University of Leicester

http://www2.le.ac.uk/departments/biology/people/mallon


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