
Dear All and Nico,

Have anyone be able to do the counting from C.elegans?

Here is what I tried but result in no luck:
1) bam was generate from tophat1.4.1, with ucsc ce6 as reference. For 
easyRNASeq counting, the *.rda was used as reference (see the code below)
obj <- fetchAnnotation(new('RNAseq',
                            organismName="Celegans"
                            ),
                        method="biomaRt")
gAnnot <- genomicAnnotation(obj)
save(gAnnot,file="ensemble_gAnnot_hsa.rda")
Here is the error message:
 > rnaSeq=easyRNASeq(filesDirectory="/cluster/easyrnaseq",
+ #organism="Celegans",
+ organism="Celegans",
+ chr.sizes=chr.sizes,
+ readLength=50L,
+ annotationMethod="rda",
+ annotationFile="/cluster/database/gtf/cel/ensemble_gAnnot_cel.rda",
+ #annotationMethod="biomaRt",
+ format="bam",
+ outputFormat="RNAseq",
+ gapped=TRUE,#for tophat bam
+ normalize=TRUE,
+ filenames="ucsc.bam",
+ count="genes",
+ summarization="geneModels",
+ validity.check=FALSE,
+ nbCore=5
+ )
Checking arguments...
Fetching annotations...
Computing gene models...
Summarizing counts...
Processing ucsc.bam
Error in aggregate.data.frame(as.data.frame(x), ...) :
   no rows to aggregate
In addition: Warning messages:
1: In easyRNASeq(filesDirectory = "/cluster/easyrnaseq",  :
   There are 8526 synthetic exons as determined from your annotation 
that overlap! This implies that some reads will be counted more than 
once! Is that really what you want?
2: In min(match(uniqueClasses, classOrder)) :
   no non-missing arguments to min; returning Inf
3: In SimpleAtomicList(result) : NAs introduced by coercion
2) I tried the same code with human bam file (except the species was 
changed to human), it works.
3) I also re-did the alignment with ensemble genome references, but 
still failed while counting.
4) The annotation files was changed to "gtf", and the gtf was download 
from ensemble. again, it is not working.
5) The organism was changed to "custom", still not working.

All hints on this issue were very much appreciated.

--
All The Best,
Yonggan
------------------------------------------------------------------------------------------
Yonggan Wu, M.S.
Bioinformatic Scientist
Ocean Ridge Biosciences LLC
10475 Riverside Drive Suite 1
Palm Beach Gardens, FL 33410
Phone : 561-223-3152
Fax : 561-740-8710
yongganw@oceanridgebio.com <mailto:yongganw@oceanridgebio.com>
http://www.oceanridgebio.com <http://www.oceanridgebio.com/>


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