question with read.HTSeqCounts function in DEXSeq package

[bioliyezhang]

Hi,

recently I tried to use DEXSeq package to estimate the differentially incorporated exons. I had a problem when I am trying to read the files.

My pipeline is:
1. annotation_DEXSeq<-"/Users/lxz/Dropbox/protocol/3-database/human_genome/Homo_sapiens.GRCh37.65.DEXSeq.gff"
2. file_path_ALK<-"/Users/lxz/Dropbox/Lab_Resource/RNAseq/1-data-processing/10-splicing/2-DEXSeq/1-DEXSeq-results"
3. samples_DHL=data.frame(condition=c(rep("control",3),rep("treated",3)),row.names=dir(file_path_ALK,pattern="DEXSeq_DHL.txt"),stringsAsFactors=TRUE, check.names=FALSE)
> samples_DHL
condition
control01_DEXSeq_DHL.txt control
control02_DEXSeq_DHL.txt control
control03_DEXSeq_DHL.txt control
treated01_DEXSeq_DHL.txt treated
treated02_DEXSeq_DHL.txt treated
treated03_DEXSeq_DHL.txt treated

4. DHL_DEXSeq=read.HTSeqCounts(countfiles=file.path(file_path_ALK,rownames(samples_DHL)),design=samples_DHL,flattenedfile=annotation_DEXSeq)

the error message is:
"Error in order(aggregates$gene_id) :
unimplemented type 'list' in 'orderVector1"

any idea what is the problem and how to fix it?
Thanks.


R environment is listed here.
> sessionInfo()
R version 2.15.1 (2012-06-22)
Platform: x86_64-apple-darwin9.8.0/x86_64 (64-bit)

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

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

other attached packages:
[1] DEXSeq_1.4.0 Biobase_2.18.0 BiocGenerics_0.4.0 BiocInstaller_1.8.3

loaded via a namespace (and not attached):
[1] biomaRt_2.14.0 hwriter_1.3 RCurl_1.95-3 statmod_1.4.16 stringr_0.6.2 tools_2.15.1
[7] XML_3.95-0.1

[Simon Anders]

This looks like something is wrong with your flattened annotation file. Have you had a look inside it to check whether it looks alright? Does 'read.HTSeqCounts' work if you don't pass the annotation file?

[bioliyezhang]

This looks like something is wrong with your flattened annotation file. Have you had a look inside it to check whether it looks alright? Does 'read.HTSeqCounts' work if you don't pass the annotation file?

Hi, Simon:

thanks. The error message disappear when I did not include the annotation file in the read.HTSeqCounts. I will check whether the downstream analysis work or not.

I also check the number of the line in reference and output.
Length of reference is 619124; length of output is 567380.
So a follow-up question, does this mean if reference and output are not in the same length, then I should not pass annotation file to read.HTSeqCounts?
Thanks.