[BioC] it is strange that I cannot get the fpkm by using easyRNASeq

[Nicolas Delhomme]

Dear Fuyan,

No worries.

You seem to have put the finger on a bug, I'll check that out. A new version of R and Bioc is available and any change I would do would go into that new version (will be 1.6.1 then).

As we discussed offline, remember that easyRNASeq won't give you FPKM sensus-stricto. What you really get are RPKMs.

Nico

---------------------------------------------------------------
Nicolas Delhomme

Nathaniel Street Lab
Department of Plant Physiology
Umeå Plant Science Center

Tel: +46 90 786 7989
Email: nicolas.delhomme at plantphys.umu.se
SLU - Umeå universitet
Umeå S-901 87 Sweden
---------------------------------------------------------------

On Apr 9, 2013, at 11:13 AM, Fuyan [guest] wrote:

> 
> Dear Nico,
> 
> Sorry to disturb you again.
> 
> I am trying to use easyRNASeq to calculate fpkm values.
> 
> 
> 
> I used the sample from the vignette, but all I get are "NA".
> 
> Below are the details:
> 
> 
> 
> 
>> count.table <- easyRNASeq(system.file(
> +  "extdata",
> +  package="RnaSeqTutorial"),
> +  organism="Dmelanogaster",
> +  readLength=30L,
> +  annotationMethod="rda",
> +  annotationFile=system.file(
> +  "data",
> +  "gAnnot.rda",
> +  package="RnaSeqTutorial"),
> +  count="exons",
> +  filenames=c("ACACTG.bam", "ACTAGC.bam",
> +  "ATGGCT.bam", "TTGCGA.bam"),
> +  normalize=TRUE
> +  )
> Checking arguments... 
> Fetching annotations... 
> Summarizing counts... 
> Processing ACACTG.bam 
> Processing ACTAGC.bam 
> Processing ATGGCT.bam 
> Processing TTGCGA.bam 
> Preparing output 
> Normalizing counts 
> Warning messages:
> 1: In easyRNASeq(system.file("extdata", package = "RnaSeqTutorial"),  :
>   There are 50573 features/exons defined in your annotation that overlap! This implies that some reads will be counted more than once! Is that really what you want?
> 2: In easyRNASeq(system.file("extdata", package = "RnaSeqTutorial"),  :
>   You enforce UCSC chromosome conventions, however the provided annotation is not compliant. Correcting it.
> 3: In fetchCoverage(rnaSeq, format = format, filename = filename, filter = filter,  :
>  You enforce UCSC chromosome conventions, however the provided alignments are not compliant. Correcting it.
> 4: In fetchCoverage(rnaSeq, format = format, filename = filename, filter = filter,  :
>  You enforce UCSC chromosome conventions, however the provided alignments are not compliant. Correcting it.
> 5: In fetchCoverage(rnaSeq, format = format, filename = filename, filter = filter,  :
>   You enforce UCSC chromosome conventions, however the provided alignments are not compliant. Correcting it.
> 6: In fetchCoverage(rnaSeq, format = format, filename = filename, filter = filter,  :
>  You enforce UCSC chromosome conventions, however the provided alignments are not compliant. Correcting it.
> 
> 
> 
> 
>> head(count.table)
>          ACACTG.bam ACTAGC.bam ATGGCT.bam TTGCGA.bam
> CG11023:1         NA         NA         NA         NA
> CG11023:2         NA         NA         NA         NA
> CG11023:3         NA         NA         NA         NA
> CG2671:1          NA         NA         NA         NA
> CG2671:2          NA         NA         NA         NA
> CG2671:3          NA         NA         NA         NA
> 
> 
> 
> while the easyRNASeq works well without normalize=TRUE:
>> head(count.table)
>          ACACTG.bam ACTAGC.bam ATGGCT.bam TTGCGA.bam
> CG11023:1          0          0          0          0
> CG11023:2          0          0          0          0
> CG11023:3          0          0          0          1
> CG2671:1           0          0          0          0
> CG2671:2           1          0          0          1
> CG2671:3          13          8         11         12
> 
> 
> 
> 
> 
> 
> I do not know what is wrong with my program.
> 
> When I am trying to use my data, I  also only get "NA" in the result.
> 
> -- output of sessionInfo(): 
> 
>> sessionInfo() 
> R version 2.15.1 (2012-06-22)
> Platform: i386-pc-mingw32/i386 (32-bit)
> 
> locale:
> [1] LC_COLLATE=English_United States.1252  LC_CTYPE=English_United States.1252    LC_MONETARY=English_United States.1252
> [4] LC_NUMERIC=C                           LC_TIME=English_United States.1252    
> 
> attached base packages:
> [1] parallel  stats     graphics  grDevices utils     datasets  methods   base     
> 
> other attached packages:
> [1] BSgenome.Dmelanogaster.UCSC.dm3_1.3.19 RnaSeqTutorial_0.0.11                  easyRNASeq_1.4.2                      
> [4] ShortRead_1.16.4                       latticeExtra_0.6-24                    RColorBrewer_1.0-5                    
> [7] Rsamtools_1.10.2                       DESeq_1.10.1                           lattice_0.20-14                       
> [10] locfit_1.5-8                           BSgenome_1.26.1                        GenomicRanges_1.10.7                  
> [13] Biostrings_2.26.3                      IRanges_1.16.6                         edgeR_3.0.8                           
> [16] limma_3.14.4                           biomaRt_2.14.0                         Biobase_2.18.0                        
> [19] genomeIntervals_1.14.0                 BiocGenerics_0.4.0                     intervals_0.14.0                      
> 
> loaded via a namespace (and not attached):
> [1] annotate_1.36.0      AnnotationDbi_1.20.7 bitops_1.0-5         DBI_0.2-5            genefilter_1.40.0    geneplotter_1.36.0   grid_2.15.1         
> [8] hwriter_1.3          RCurl_1.95-4.1       RSQLite_0.11.2       splines_2.15.1       stats4_2.15.1        survival_2.37-4      tools_2.15.1        
> [15] XML_3.95-0.2         xtable_1.7-1         zlibbioc_1.4.0 
> 
> --
> Sent via the guest posting facility at bioconductor.org.