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Old 02-24-2014, 07:06 AM   #1
super0925
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Location: UK

Join Date: Feb 2014
Posts: 206
Question Error in htseq count "Please provide two arguments"

Hi All
I am a rookie in RNA-seq, and I have some problems.
I have tried the pipeline of Tuxedo (Tophat-Cufflinks-Cuffdiff) to analysis the data.
Now I want to change some other pipelines (DESeq or edgeR, etc) to compare the performance.
However, as you know, these R-based package need the software htseq-count to generate the annotated count matrix.
So I use this command but get the error:
htseq-count -m intersection-strict -s no accepted_hits.sam merged.gtf>subset.counts
where accepted_hits.sam is file convert from accepted_hits.bam which is generated by Tophat. merged.gtf is the merged annotation generated by Cufflinks-Cuffmerge.
Is something wrong I did?
Or I need to use the reference genome:
genes.gtf but not merged.gtf
which is GTF/GFF file I need to use? reference genome or assembled transcripts? Thank you!

Last edited by super0925; 02-25-2014 at 03:37 AM.
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Old 02-25-2014, 03:37 AM   #2
super0925
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Location: UK

Join Date: Feb 2014
Posts: 206
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Quote:
Originally Posted by super0925 View Post
Hi All
I am a rookie in RNA-seq, and I have some problems.
I have tried the pipeline of Tuxedo (Tophat-Cufflinks-Cuffdiff) to analysis the data.
Now I want to change some other pipelines (DESeq or edgeR, etc) to compare the performance.
However, as you know, these R-based package need the software htseq-count to generate the annotated count matrix.
So I use this command but get the error:
htseq-count -m intersection-strict -s no accepted_hits.sam merged.gtf>subset.counts
where accepted_hits.sam is file convert from accepted_hits.bam which is generated by Tophat. merged.gtf is the merged annotation generated by Cufflinks-Cuffmerge.
Is something wrong I did?
Or I need to use the reference genome:
genes.gtf but not merged.gtf
Thank you!
which is GTF/GFF file I need to use? reference genome or assembled transcripts? Thank you!
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Old 02-25-2014, 10:42 PM   #3
Simon Anders
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Location: Heidelberg, Germany

Join Date: Feb 2010
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Are you sue the command you typed is exactly as you posted it here? The error message means that you did not specify two files -- it is generated before the script actually looks into the files, so it has nothing to do with their content.
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Old 02-26-2014, 12:48 AM   #4
super0925
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Location: UK

Join Date: Feb 2014
Posts: 206
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Quote:
Originally Posted by Simon Anders View Post
Are you sue the command you typed is exactly as you posted it here? The error message means that you did not specify two files -- it is generated before the script actually looks into the files, so it has nothing to do with their content.
I am sure.
But when I tried the genes.gtf, it works. but merged.gtf, it is not working.
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