Question: HTSEQ count error
gravatar for gilberjr
5 months ago by
gilberjr0 wrote:


This is my first rna-seq data analysis project and I am using Galaxy to analyze differential expression on my RNA-seq data comparing 4 treatments. So far I have successfully trimmed my raw data using Trimmomatic, and aligned my reads using HISAT2. Now I am trying to use the htseq-count and was successful with my first few runs, but am now getting an error message for some files. I have tried re-running these several times and still end up with a failed run.

Should I abandon htseq and use another count tool or can I still use the data (that did not fail), discarding the failed files, to continue with my research?


rna-seq galaxy • 236 views
ADD COMMENTlink modified 5 months ago by Jennifer Hillman Jackson23k • written 5 months ago by gilberjr0

What error message are you getting and from which tool and version (htseq_count)? Are you working at

Post the error itself back as a comment with the format as "Code Sample" or share a link to an external site where formatting is preserved, such as a Gist.

The problem is likely a data format/content issue with the fastq input, or htseq_count usage, but let's review the error and decide if that is where to look for the problem. Discarding data would probably be a mistake unless it is totally unusable and it doesn't impact your primary experimental goals.

Thanks! Jen, Galaxy team

ADD REPLYlink modified 5 months ago • written 5 months ago by Jennifer Hillman Jackson23k

I am using htseq-count at, (Galaxy Version 0.6.1galaxy3).

Code Sample: Fatal error: Unknown error occured 83877 GFF lines processed. Error occured when reading beginning of SAM/BAM file.

ADD REPLYlink written 5 months ago by gilberjr0
gravatar for Jennifer Hillman Jackson
5 months ago by
United States
Jennifer Hillman Jackson23k wrote:


Thanks for sending in the extra information.

Check your BAM/SAM inputs - do they have content other than the header (indicating that mapping failed)? If SAM, do these even have a header (this is required by the tool).

To troubleshooting mapping, make sure the correct target database was selected and that the input fastq data is in fastqsanger format. Sorting also sometimes helps resolve errors but not for this specific case that I know of. More help details here that resolve the majority reported issues:

If you still cannot determine the problem after reviewing, a bug report can be sent in. Please include a link to this Biostars post in the comments so we can associate the two and be sure to not to delete the inputs or error datasets (or undelete them if you need to).

Thanks, Jen, Galaxy team

ADD COMMENTlink modified 5 months ago • written 5 months ago by Jennifer Hillman Jackson23k
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