htseq-count crashed again and again

Question: htseq-count crashed again and again

5 months ago by

htseq-count crashed again and again Usegalaxy.org)

Running output file from HiSat2, Runs for 20 min and give message:
error
An error occurred with this dataset:
Fatal error: Unknown error occured
[bam_sort_core] merging from 62 files and 1 in-memory blocks...
100000 GFF lines processed.
200000 GFF lines processed.
300000 GFF lines processed.
400000 GFF lines processed.
500000 GFF lines processed.
600000 GFF line

Whats wrong? Space is OK -50% z

count memory rna-seq htseq-count htseq • 293 views

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modified 5 months ago • written 5 months ago by andrewzlobin • 20

5 months ago by

Jennifer Hillman Jackson ♦ 25k

United States

Jennifer Hillman Jackson ♦ 25k wrote:

Hello,

There are a few problems:

This tool does not accept data in GFF3 format. It does accept GTF format and GFF format that includes key attributes of strict GTF format.
The reference annotation in now an error state due to the failed conversion to other datatypes.
The reference annotation is based on the Ensembl genome build/version, which has chromosome naming mismatch problems versus the UCSC rn6 genome build/version used for mapping.

Choices:

All inputs must be based on the same genome build/version for errors or unexpected results can occur.
Find a GTF version of the annotation based on rn6. Many data sources will provide both. For UCSC's rn6 genome build/version, iGenomes is one provider for reference annotation. Download the tar archive locally, uncompress, and Upload just the genes.gtf dataset to Galaxy. https://support.illumina.com/sequencing/sequencing_software/igenome.html
The tool gffread can convert GFF3 to GFT, but the original GFF3 still needs to be based on the same genome build/version. The one you have currently will not work.

All of the above is covered in the Support FAQs: https://galaxyproject.org/support/#troubleshooting

Galaxy tutorials, including those for RNA-seq: https://galaxyproject.org/learn/

Thanks! Jen, Galaxy team

ADD COMMENT • link written 5 months ago by Jennifer Hillman Jackson ♦ 25k

5 months ago by

andrewzlobin • 20

andrewzlobin • 20 wrote:

i am very grateful, thanks a lot. I am doing what you recommend. Just another question, for HISAT I was using the fast file. Is it supposed to be also from UCSC? I don't remember where I downloaded it.

Thanks Andrei

ADD COMMENT • link written 5 months ago by andrewzlobin • 20

Cross-posted here: https://biostar.usegalaxy.org/p/28491/

ADD REPLY • link written 5 months ago by Jennifer Hillman Jackson ♦ 25k

5 months ago by

andrewzlobin • 20

andrewzlobin • 20 wrote:

Dear Jennifer, I uploaded Rattus_norvegicus_UCSC_rn6.tar, Htseq-count still doesn't work. ???

Feature-count does work!!! with this dataset!!!! Fantastic! I hope this will sort out problem.

Thanks a lot Andrew

ADD COMMENT • link written 5 months ago by andrewzlobin • 20

5 months ago by

andrewzlobin • 20

andrewzlobin • 20 wrote:

Great I was able to run featurecounts output files on DeSeq2. All work.fantastic Thanks a lot.

ADD COMMENT • link written 5 months ago by andrewzlobin • 20

5 months ago by

andrewzlobin • 20

andrewzlobin • 20 wrote:

With the most resent rat annotation file I was able to run HTseq-count on my data. But after that when I try to run output files on DESeq2 it crashes:" Fatal error: An undefined error occurred, please check your input carefully and contact your administrator. Error in row.names<-.data.frame(*tmp*, value = value) : duplicate 'row.names' are not

What's the reasons? Thanks Andrew

ADD COMMENT • link written 5 months ago by andrewzlobin • 20

5 months ago by

andrewzlobin • 20

andrewzlobin • 20 wrote:

Sorry I found the problem. Thanks Andrew

ADD COMMENT • link written 5 months ago by andrewzlobin • 20

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