[dpryan]

You might just try:

rawcounts <- read.delim("counts.txt", header=T, row.names=1)

[huma Asif]

can you help me how i can get rid of 1,2,3, in first column
> head(rawcounts)
Gene.ID A B
1 WASH7P 1 0
2 FAM138A 0 0
3 FAM138F 0 0
4 OR4F5 0 0
5 LOC100132062 0 0
6 LOC100132287 0 0
i tried
rawcounts <- read.delim("counts.txt", header=TRUE, sep="/t", row.names="Gene.ID")
but it doesnot work

[huma Asif]

thank you dpryan

[dpryan]

That ID corresponds to whatever you counted on in your annotation file. If the corresponding names are in the file then just load it (see the GenomicFeatures package) and use that. Alternatively, there are annotation packages for many organisms, so just load the appropriate one in R and use it.

[huma Asif]

while its going let me explain you a problem about DESeq
i tried to use input file with genenames but it was giving me this error
Error in round(countData) : non-numeric argument to mathematical function

so what i did in analysis i cut the column with gene name
now problem is the interpretation of result as in output i have id and i am confused how to combine this info with my gene
i want to attach my count file but here is no option to attach



output (total)
id baseMean baseMeanA baseMeanB foldChange log2FoldChange pval padj
18 32.80688778 4.323701761 61.2900738 14.17537036 3.825314524 0.034361637 0.836923637

output to see just significant
id baseMean baseMeanA baseMeanB foldChange log2FoldChange pval padj
15959 3564.773938 16.43006669 7113.11781 432.9329846 8.757999912 8.02E-20 1.39E-15

problem here is what this id is telling me and how i am going to combine with gene names

please do reply atleast this DESeq output will give me an idea to compare with others

[dpryan]

Normally you create a character vector containing filenames that you would use (it's usually best to just make a CSV file with all of this and then load it). Otherwise, yes, you have to type it all by hand.

[huma Asif]

thank you i read all that but do i need to write names of my count file or path where my files are my current diectory is where my gff and count files are

[dpryan]

Please read

help(DEXSeqDataSetFromHTSeq)

You'll see that the countFiles are the files containing counts produced by dexseq_count.py.

[huma Asif]

please help me in creating
DEXSeqDataSet
i am confused what shall i give in place of countFiles
where do i specify my files in this code



dxd = DEXSeqDataSetFromHTSeq(
countFiles,
sampleData=sampleTable,
design= ˜ sample + exon + condition:exon,
flattenedfile=flattenedFile )

[huma Asif]

> ecs <- read.HTSeqCounts(
+ sampleTable$countFile,
+ sampleTable,
+ flattenedfile="gene.gff")
Error in checkAtAssignment("character", "annotationFile", "character") :
‘annotationFile’ is not a slot in class “character”
In addition: Warning message:
'newExonCountSet' is deprecated.
Use 'DEXSeqDataSet' instead.
See help("Deprecated")

[huma Asif]

i am using
> packageVersion("DEXSeq")
[1] ‘1.10.8’

[dpryan]

What version of DEXseq are you using? That entire method has been deprecated. Anyway, try:

flattenedfile="gene.gff"

instead of just "gene.gff".

[huma Asif]

thanks i downloaded gtf from ucsc

well i am following instruction in
Inferring differential exon usage in RNA-Seq data with the DEXSeq package
done with
dexseq_prepare_annotation.py script and
python /path/to/library/DEXSeq/python_scripts/dexseq_count.py
Dmel_flattenend.gff untreated1.sam untreated1.counts

done with > sampleTable
countFile condition libType
A A.counts control single-end
B B.counts patient single-end


need help here
ecs <- read.HTSeqCounts(
+ sampleTable$countFile,
+ sampleTable,
+ "gene.gff" )

getting this error
Error: unexpected string constant in:
"sampleTable
"gene.gff""

[dpryan]

Regarding the Ensembl annotation, you could just use sed or awk to change the chromosome names (note that the mitochondria are chrM in UCSC and MT in Ensembl, so just appending "chr" won't fix everything).

Regarding the DEXSeq error, it would be helpful to see the command issued and the exact error message.

[huma Asif]

for DEXSeq i downloaded Homo_sapiens.gtf from UCSC but it terminate everytime on chr15
from ensembl it is downloaded successfully but now prob is chr in bam and 1,2,... chromosome notation
can you guide how to make it compatible