Fatal error: Exit code 1 () when using SAM FASTA Index Builder.

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Hi,

I am relatively new to using Galaxy, and I am attempting to try to eventually align my paired RNAseq data using STAR. I am currently running Galaxy (version 19.09) from a personal computer using a University open local server. I have successfully used the tool ‘Create DBKey and Reference Genome’ using the existing DBkey assigned as Mouse Dec. 2011 (GRCm38/mm10) (mm10) sourced from UCSC (with mm10 inputted into the field of ‘UCSC’s DBKEY for source FASTA’).

However, I have now come across a Fatal error: Exit code 1 () when I have tried to use the data_manager_sam_fasta _Index_builder tool using the DBKey I have created from the previous step (i.e mm10). The Dataset Information and standard error generated by the tool is below;

Dataset information:

Name: SAM FASTA index

Created: Mon Mar 2 19:47:01 2020 (UTC)
Filesize: 2.8 KB
Dbkey:?
Format:data_manager_json

Standard error:

Traceback (most recent call last):
File “/var/www_remote/galaxy.pharm.nottingham.ac.uk_19.09/database/shed_tools/toolshed.g2.bx.psu.edu/repos/devteam/data_manager_sam_fasta_index_builder/f8418a1bf7d0/data_manager_sam_fasta_index_builder/data_manager/data_manager_sam_fasta_index_builder.py”, line 100, in
main()
File “/var/www_remote/galaxy.pharm.nottingham.ac.uk_19.09/database/shed_tools/toolshed.g2.bx.psu.edu/repos/devteam/data_manager_sam_fasta_index_builder/f8418a1bf7d0/data_manager_sam_fasta_index_builder/data_manager/data_manager_sam_fasta_index_builder.py”, line 92, in main
data_table_name=options.data_table_name or DEFAULT_DATA_TABLE_NAME
File “/var/www_remote/galaxy.pharm.nottingham.ac.uk_19.09/database/shed_tools/toolshed.g2.bx.psu.edu/repos/devteam/data_manager_sam_fasta_index_builder/f8418a1bf7d0/data_manager_sam_fasta_index_builder/data_manager/data_manager_sam_fasta_index_builder.py”, line 32, in build_sam_index
assert os.path.exists(fasta_filename), ‘FASTA file “%s” is missing, cannot build samtools index.’ % fasta_filename
AssertionError: FASTA file “/users2/galaxy.pharm.nottingham.ac.uk/tool-data/mm10/seq/mm10.fa,/var/www_remote/galaxy.pharm.nottingham.ac.uk/tool-data/mm10/seq/mm10.fa,/var/www_remote/galaxy.pharm.nottingham.ac.uk_19.09/tool-data/mm10/seq/mm10.fa” is missing, cannot build samtools index.

There is most likely something really simple in which I am doing wrong, but I can’t seem to find any suggestions in previous forums which can solve this issue.

I am hoping to eventually follow previous guides of indexing this reference genome prior to using the STAR alignment tool on my server using Data Manager tools listed in sequence below;

  1. SAM indexer.
  2. Picard Indexer.
  3. 2bit (twoBit) indexer.

Any suggestions/help will be greatly appreciated.

Many thanks,
Steven.

2

I don’t know much about this tool or method but look at the error.

AssertionError: FASTA file “/users2/galaxy.pharm.nottingham.ac.uk/tool-data/mm10/seq/mm10.fa,/var/www_remote/galaxy.pharm.nottingham.ac.uk/tool-data/mm10/seq/mm10.fa,/var/www_remote/galaxy.pharm.nottingham.ac.uk_19.09/tool-data/mm10/seq/mm10.fa”

This:

“/users2/galaxy.pharm.nottingham.ac.uk/tool-data/mm10/seq/mm10.fa,/var/www_remote/galaxy.pharm.nottingham.ac.uk/tool-data/mm10/seq/mm10.fa,/var/www_remote/galaxy.pharm.nottingham.ac.uk_19.09/tool-data/mm10/seq/mm10.fa”

Does not look like a fasta file to me. It are multiple file paths that are separated by a comma. Maybe you know where this comes from.

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3

Yup, the entries in all_fasta.loc are wrong, this can happen when running the data manager multiple times for the same dbkey.

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