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Old 11-19-2010, 02:26 PM   #1
nimmi
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Default understanding HTSeq counts

Hi,

I ran HTSeq on a *.sam file using RefSeq genes GTF file. I got counts for each transcript and would like to understand how exactly these counts were generated. Does HTSeq count a read whenever it overlaps with an exon and then takes a sum/average of all these exons across a transcript? Or, is there some other complicated procedure to merge the counts of all the exons in a transcript?

Thank you very much for your response.

Nirmala
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Old 11-19-2010, 03:39 PM   #2
shurjo
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Quote:
Originally Posted by nimmi View Post
Hi,

I ran HTSeq on a *.sam file using RefSeq genes GTF file. I got counts for each transcript and would like to understand how exactly these counts were generated. Does HTSeq count a read whenever it overlaps with an exon and then takes a sum/average of all these exons across a transcript? Or, is there some other complicated procedure to merge the counts of all the exons in a transcript?

Thank you very much for your response.

Nirmala
This is a question I have also been meaning to ask. I doubt that the average would be useful here. I don't know enough Python to read the code, but maybe the authors of the software will have an answer for us here?
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Old 11-23-2010, 07:26 AM   #3
Simon Anders
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htseq-count is a rather simple script. First of all, it does not attempt to tease apart isoforms (transcripts), it only counts for genes. So, if a read overlaps with one or more exons of a gene, it is counted for this gene. If it overlaps with exons from more then one gene, it it counted as ambiguous, i.e, for neither of the genes. The precise definition of "overlap" can be adjusted, see the figure at http://www-huber.embl.de/users/ander...doc/count.html .

Simon
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Old 11-27-2010, 07:24 PM   #4
ecofriendly
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hello,

This is a great forum and I'm learning a lot here. I would really appreciate some help troubleshooting HTSeq.

I've installed everything correctly in the Python window, I think, since I get no error messages. From the command line, I then type:

python -m HTSeq.scripts.count -q <sam.file> <gtf.file>

Since I'm running the script on quiet mode, I get the following output:

no_feature 0
ambiguous 0
too low aQual 0
not aligned 0

I tried writing the countsTable to a file, by adding a "> countsTable.txt" to the end of the above, but this text file contains the exact same info as was printed above.

There's nothing wrong with either the SAM file created by TopHat and Samtools, or with the GTF file, as I've worked with both of them successfully in other programs.

Thanks for any help!!

elena

Last edited by ecofriendly; 11-27-2010 at 07:28 PM.
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