# Continuing the discussion from \[RSEM prepare reference tool does not produce valid reference files\] **URL:** \ **Category:** usegalaxy.eu support\ **Tags:** troubleshooting, transcriptomics, rna-seq\ **Created:** [May 4, 2023, 9:53am UTC](https://help.galaxyproject.org/t/continuing-the-discussion-from-rsem-prepare-reference-tool-does-not-produce-valid-reference-files/10006 "2023-05-04T09:53:46Z")\ **Posts on this page:** 3\ **Page:** 1
**Author:** ![qcsciphi](https://avatars.discourse-cdn.com/v4/letter/q/c5a1d2/32.png) [@qcsciphi](https://help.galaxyproject.org/u/qcsciphi)\ **Post date:** [May 4, 2023, 9:53am UTC](https://help.galaxyproject.org/t/continuing-the-discussion-from-rsem-prepare-reference-tool-does-not-produce-valid-reference-files/10006/1 "2023-05-04T09:53:47Z")
Continuing the discussion from [RSEM prepare reference tool does not produce valid reference files](https://help.galaxyproject.org/t/rsem-prepare-reference-tool-does-not-produce-valid-reference-files/8109): Dear specialists, I wold like to ask your help and attention on “RSEM prepare reference” tool which fails to prepare RSEM reference both on “transcript fasta” and on “reference genome and GTF” as reference transcript source. > tool finishes job with empty output with the warning below on “transcript fasta”; RSEM\_Ref\_Gencode43\_CDS WARNING:galaxy.model:Datatype class not found for extension ‘rsem\_ref’ > the tool fails on “reference genome and GTF” with a standard error > I created a bug report from this failing job. Would you please suggest how to proceed to generate RSEM reference on CDS with the “transcript fasta” option? Please find the link to the history below; > **[Galaxy](https://usegalaxy.eu/published/history?id=c0c0124d45d68034)** > > Galaxy is a community-driven web-based analysis platform for life science research. Kind regards, Serdar ---
**Author:** ![jennaj](https://sea2.discourse-cdn.com/flex020/user_avatar/help.galaxyproject.org/jennaj/32/27_2.png) [@jennaj](https://help.galaxyproject.org/u/jennaj)\ **Post date:** [May 4, 2023, 9:07pm UTC](https://help.galaxyproject.org/t/continuing-the-discussion-from-rsem-prepare-reference-tool-does-not-produce-valid-reference-files/10006/2 "2023-05-04T21:07:22Z")
Hi @qcsciphi The message in one of the red error datasets: > RSEM\_Ref\_Gencode43\_only > Cannot extract transcript ENST00000373020.9’s sequence from chromosome ENSG00000000003.15, whose information might not be provided! Please check if the chromosome directory is set correctly or the list of chromosome files is compl (ete) ENST00000373020.9 is a transcript ID ENSG00000000003.15 is a gene ID, but the tool thinks it is where a chromosome should be specified? This was another error: > RSEM\_Ref\_Ensemble109\_only > .gtf file might be corrupted! > Stop at line : 1 ensembl\_havana gene 1471765 1497848 . + . gene\_id “ENSG00000160072”; gene\_version “20”; gene\_name “ATAD3B”; gene\_source “ensembl\_havana”; gene\_biotype “protein\_coding”; 1 is a chromosome ENSG00000160072 is a gene ID and that file has no transcript specified on that line. What to modify or check: 1. Each input area on the form expects data in specific formats, and with the matching datatype assigned. 2. If you are not sure what datatypes are expected, see [fastq unavailable -- Tool does not recognize inputs? How to check why](https://help.galaxyproject.org/t/fastq-unavailable-tool-does-not-recognize-inputs-how-to-check-why/8925) 3. Double check that the content of those data follow the datatype specification. 4. Check across files for consistency. Tools are literal when making matches between important identifiers. - Meaning, if one file has an identifier, then other files should have that exact identifier, not a variation. - Example of a mismatch: - Another example of a mismatch: Some FAQs contain datatype format/content help, and this whole FAQ page can be browser-keyword searched. [https://training.galaxyproject.org/training-material/faqs/galaxy/](https://training.galaxyproject.org/training-material/faqs/galaxy/) - [Working with very large fasta datasets](https://training.galaxyproject.org/training-material/faqs/galaxy/datasets_working_with_fasta.html) - [Working with GFF GFT GTF2 GFF3 reference annotation](https://training.galaxyproject.org/training-material/faqs/galaxy/datasets_working_with_reference_annotation.html) - [Mismatched Chromosome identifiers and how to avoid them](https://training.galaxyproject.org/training-material/faqs/galaxy/datasets_chromosome_identifiers.html) Please give these a review, then adjust your data. ---
**Author:** ![jennaj](https://sea2.discourse-cdn.com/flex020/user_avatar/help.galaxyproject.org/jennaj/32/27_2.png) [@jennaj](https://help.galaxyproject.org/u/jennaj)\ **Post date:** [December 20, 2023, 1:00am UTC](https://help.galaxyproject.org/t/continuing-the-discussion-from-rsem-prepare-reference-tool-does-not-produce-valid-reference-files/10006/3 "2023-12-20T01:00:24Z")
Closed on December 20, 2023, 1:00am UTC