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@badribio-12724
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suppressPackageStartupMessages( library( "DEXSeq" ) )
setwd("pathto/htseq")
inDir = getwd()
countFiles = list.files(inDir, pattern = "htseq.txt$", full.names = TRUE)
flattenedFile = ("pathto/ensembl/Mus_musculus.NCBIM37.67.gtf")
mouseTable = data.frame(
row.names = c("S75","S76","S77","S72","S73","S74"),
condition = c("WT","WT","WT","MT","MT","MT"),
libType = c("paired-end","paired-end","paired-end","paired-end","paired-end","paired-end"))
mouseTable
dxd = DEXSeqDataSetFromHTSeq(countFiles,sampleData = mouseTable,design = ~ sample + exon + condition:exon, flattenedfile = flattenedFile)
37996 ./76.htseq.txt
37996 ./72.htseq.txt
37996 ./77.htseq.txt
37996 ./75.htseq.txt
37996 ./73.htseq.txt
37996 ./74.htseq.txt
Number of lines are same in all the count files and none of them have any headers. I used the same gtf file for htseq.
not sure where am I going wrong due to which I get this error
Error in FUN(X[[i]], ...) : subscript out of bounds
Thank you in anticipation.
Hi. Are you using the python scripts dexseq_prepare_annotation.py and dexseq_count.py) that come with the DEXSeq package?
let me walk you through my steps may be you could please point out where i went wrong,
1) Bam files processed using Htseq count with parameters with parameters: --mode intersection-strict --minaqual 1 --stranded reverse --type exon --idattr gene_id using gtf (not gff) from mm9.37.67(ensembl)
2) load the output for 6 samples using "DEXSeqDataSetFromHTSeq" <- I am stuck here for now
3) also if i supply the gff from dexseq_prepare_annotation.py while using htseq, i get "Warning: No features of type 'exon' found" as an error.
4) I also have the results from dexseq_count.py as well as explained in the DEXSeq manual, but the paper I am referring to uses htseq counts.
Thank you, for replying!!! really appreciate it.
I think I did not understand the reason why you are not using the scripts from dexseq. But if you want to use htseq instead to count on exons, you might want to read the documentation from the --type and --idattr parameters.
Also, notice that the function DEXSeqDataSetFromHTSeq is expecting the output from the dexseq scripts, so it is likely that it won't work with the output of htseq-count. Nevertheless, you can still use the DEXSeqDataSet function to create a DEXSeqDataSet object.
Alejandro
so if I am comprehending it correct although the function is DEXSeqDataSetFromHTSeq it still expects the input from dexseq_count.py? If so then could you please let me know which file would the right result to use for downstream analysis based on my sequencing type?
rna seq data paired end stranded (tophat option - fr-firststrand)
dexoptions
dexout$ tail S72.dex.reverse.txt
ENSMUSG00000093784+ENSMUSG00000009292+ENSMUSG00000093443:058 25
ENSMUSG00000093785:001 0
ENSMUSG00000093786:001 0
ENSMUSG00000093787:001 0
ENSMUSG00000093788:001 0
_ambiguous 155274
_ambiguous_readpair_position 5427516
_empty 43670128
_lowaqual 0
_notaligned 0
dexout$ tail S72.dex.txt
ENSMUSG00000093784+ENSMUSG00000009292+ENSMUSG00000093443:058 0
ENSMUSG00000093785:001 0
ENSMUSG00000093786:001 0
ENSMUSG00000093787:001 5
ENSMUSG00000093788:001 0
_ambiguous 9089
_ambiguous_readpair_position 5427516
_empty 90229017
_lowaqual 0
_notaligned 0
Many thanks
so if I am comprehending it correct although the function is DEXSeqDataSetFromHTSeq it still expects the input from dexseq_count.py?
Yes
If so then could you please let me know which file would the right result to use for downstream analysis based on my sequencing type?
Not sure I can know that. Would recommend to have a loot at your bam files in a browser to get an idea.
Snap shot of one of the sample, in igv