Hisat2 BAM file output does not include stats -- Not a default output but is an option on the tool form

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I’m not getting the stats when running my Hisat2 alignment, I’ve already followed the suggestion of checking the stdout and stderr sections but there is nothing there either. The statistics of bam.iobio tell me I have a good quality alignment, though. Can I trust those results or what should I do?

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Hi @MarioG

https://bam.iobio.io is a nice graphical summary tool for BAM/SAM alignment stats.

To output statistics directly from HISAT2, that output option needs to be set to “Yes” on the tool form. Find it nested under the section “Summary Options”. This would allow you to use MultiQC to compare multiple BAM results together in a single report.

hisat2-output-alignment-stats
hisat2-output-alignment-stats1466×370 30.4 KB

There are also other ways to generate alignment statistics – review tools under the group SAM/BAM for the choices available at usegalaxy.eu.

If you plan to go forward with differential expression analysis, after generating counts (example: Featurecounts or HT-seq count), those results can be examined with scientific content metrics with the tool QualiMap Counts QC.

Thanks!

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Those options are actually set to “Yes” already, but the summary seems to be empty (0 bytes) and although the alignment is completed, it keeps sending the following error messages: Error, fewer reads in file specified with -2 than in file specified with -1
terminate called after throwing an instance of ‘int’
(ERR): hisat2-align died with signal 6 (ABRT)
[bam_sort_core] merging from 6 files and 1 in-memory blocks…
Here a picture: image
I’m concerned this might have to do with the fact that I’m using files that have been previously trimmed. I used Headcrop on Trimmomatic to trimm off the first 10 beses as they didn’t look good on the FastQC report (per base sequence content). On the other hand, when I use files that haven’t been trimmed off, HISAT2 doesn’t send any warnings and it actually shows the statistics. What do you think? I don’t feel confident about which files I should use to do the alignment.

Thank you so much for the recommended tools, Jennifer. We’ll definitely try them as soon as we move forward on the analysis.

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Hi @MarioG

Apologies for the delay.

The message about reads not being paired is just a warning. It will not cause a mapping job using HISAT2 to fail.

If you haven’t tried a rerun yet, do that now, this may have been a cluster or server issue now resolved.

If the jobs failed again, then you are actually not getting any hits for some reason. Maybe the reads are too short – the parameters are too strict – or the wrong database was mapped against? This tool is designed to find “exact matches” when all default settings are used but the details can be adjusted under “Advanced settings”.

Hope you worked this out!

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