Problems mapping with HISAT2 & BOWTIE2

usegalaxy.org support
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#1

I’m having an issue with mapping using HISAT2 and Bowtie 2. I am trying to map my genome to a reference genome. Both programs run, but the BAM files only show is QNAME and all the other components of the table are blank. I have already gone through trimming and QC and the quality scores are all above 30.

This is example of an error in mapping stats:
Warning: skipping mate #2 of read ‘V300007992L3C001R0141268281/2’ because it was < 2 characters long Warning: skipping mate #1 of read ‘V300007992L3C001R020207830/1’ because length (1) <= # seed mismatches

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#2

Hi –

For the mapping stats, scroll down the page to find the report. The warnings at the top are about reads that are too short to map with the initial seeding criteria. You could filter out very short reads by length when doing other QA steps with Trimmomatic or choose to ignore them – they are not contributing to a failure (the runs are successful).

There does seem to be a performance problem at the Galaxy Main https://usegalaxy.org server that just started. I’m checking with our administrator to find out what is going on. Expanding/viewing datasets is just one symptom. More feedback very soon & thanks for reporting the issue!

#3

Haven’t heard back from the administrator yet but I was able to expand/view datasets again. There might have been a very heavy load on the server for a short window.

Try viewing your BAM results again if you want. The data is intact and can be used as input to downstream tools, even if the “view” function is a bit slow right now.

#4

Thank you. The run are successful, but all the other columns except QNAME are blank. So, even though it is running successfully. There is a error which is causing the other columns to be blank.

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#5

This is when viewing the BAM dataset, correct? Only the first line will display immediately. BAMs are compressed data. Viewing these is a special function in Galaxy and it takes some time to load the data up in the application. I was able to look at one of your smaller results and it took a few minutes. The first line loaded first, then the header (fairly large from the custom genome), then finally the data lines after scrolling down the page.

To view or work with the content in an uncompressed format, convert with BAM-to-SAM. To generate statistics on a BAM dataset, there are many tools – search the tool panel with the keyword “bam” and/or review the tools under the group SAM/BAM.