Hi, happy new year everyone! I tried to run v4.0.0 on an HPC with Slurm, Singularity, Nextflow v25.04. The test stub run was successful. I downloaded the references and ran COSMIC Fusionreport download on my local computer. When I run the pipeline on actual samples, it's trying to download the Fusionreport reference files again. But it fails even with 'no-cosmic' although I provide the fusionreport_ref option. It seems to find all other provided reference files because some results are generated, e.g. STAR before failure. I suspect I'm overlooking something very trivial. I may be able to solve the HPC network/proxy issue but am wondering what goes wrong. My command:

nextflow run <http://main.nf|main.nf> -profile singularity -c pte.config -params-file nf-params.json -bg > logs/test.log

My params file:

{

"input": "./samplesheet.csv",

"outdir": "./results",

"tools": "fastp,all",

"references_only": false,

"genomes_base": "/lustre01/references/RNAfusion/nf-core/GRCh38",

"genomes_ignore": false,

"fasta": "/lustre01/references/RNAfusion/nf-core/GRCh38/gencode_v46/gencode/Homo_sapiens_GRCh38_46_dna_primary_assembly.fa",

"fai": "/lustre01/references/RNAfusion/nf-core/GRCh38/gencode_v46/gencode/Homo_sapiens_GRCh38_46_dna_primary_assembly.fa.fai",

"gtf": "/lustre01/references/RNAfusion/nf-core/GRCh38/gencode_v46/gencode/Homo_sapiens_GRCh38_46.gtf",

"refflat": "/lustre01/references/RNAfusion/nf-core/GRCh38/gencode_v46/gencode/Homo_sapiens_GRCh38_46.gtf.refflat",

"rrna_intervals": "/lustre01/references/RNAfusion/nf-core/GRCh38/gencode_v46/gencode/Homo_sapiens_GRCh38_46.gtf.interval_list",

"arriba_ref_blacklist": "/lustre01/references/RNAfusion/nf-core/GRCh38/arriba/2.5.0/blacklist_hg38_GRCh38_v2.5.0.tsv.gz",

"arriba_ref_cytobands": "/lustre01/references/RNAfusion/nf-core/GRCh38/arriba/2.5.0/cytobands_hg38_GRCh38_v2.5.0.tsv",

"arriba_ref_known_fusions": "/lustre01/references/RNAfusion/nf-core/GRCh38/arriba/2.5.0/known_fusions_hg38_GRCh38_v2.5.0.tsv",

"arriba_ref_protein_domains": "/lustre01/references/RNAfusion/nf-core/GRCh38/arriba/2.5.0/protein_domains_hg38_GRCh38_v2.5.0.gff3",

"fusioncatcher_ref": "/lustre01/references/RNAfusion/nf-core/GRCh38/gencode_v46/fusioncatcher/",

"hgnc_ref": "/lustre01/references/RNAfusion/nf-core/GRCh38/hgnc/hgnc_complete_set.txt",

"hgnc_date": "/lustre01/references/RNAfusion/nf-core/GRCh38/hgnc/HGNC-DB-timestamp.txt",

"salmon_index": "/lustre01/references/RNAfusion/nf-core/GRCh38/gencode_v46/salmon",

"starfusion_ref": "/lustre01/references/RNAfusion/nf-core/GRCh38/gencode_v46/starfusion/ctat_genome_lib_build_dir",

"starindex_ref": "/lustre01/references/RNAfusion/nf-core/GRCh38/gencode_v46/star",

"fusionreport_ref": "/lustre01/references/RNAfusion/nf-core/GRCh38/fusion_report_db",

"fastp_trim":"",

"trim_tail_fusioncatcher": 50,

"read_length": 151,

"species": "homo_sapiens",

"cosmic_username": "<mailto:x@y.hu|x@y.hu>",

"cosmic_passwd": "xyz"

}

fusion_report_db

content: -rwxrwxr-x 1 kjozsi bioinfo 16 Dec 2 21:59 DB-timestamp.txt -rwxrwxr-x 1 kjozsi bioinfo 20M Dec 2 21:59 cosmic.db _-rwxrwxr-x 1 kjozsi bioinfo 0 Dec 2 21:59 fusion_report.log_ -rwxrwxr-x 1 kjozsi bioinfo 2.3M Dec 2 21:59 fusiongdb2.db -rwxrwxr-x 1 kjozsi bioinfo 3.5M Dec 2 21:59 mitelman.db I appreciate any input.

Hi, could you also post the error you encounter here?

ERROR ~ Error executing process > 'RNAFUSIONBUILD REFERENCESFUSIONREPORT_DOWNLOAD (fusionreport)' Caused by: Process

RNAFUSION:BUILD_REFERENCES:FUSIONREPORT_DOWNLOAD (fusionreport)

terminated with an error exit status (1) Command executed: fusion_report download --no-cosmic ./ mkdir fusion_report_db mv *.txt *.log *.db fusion_report_db/ cat <<-END_VERSIONS > versions.yml "RNAFUSIONBUILD REFERENCESFUSIONREPORT_DOWNLOAD": fusion_report: $(fusion_report --version | sed 's/fusion-report //') END_VERSIONS Command exit status: 1 Command output: [32mDownloading resources...[0m [32mDownloading https://storage.googleapis.com/mitelman-data-files/prod/mitelman_db.zip[0m [31mError downloading https://storage.googleapis.com/mitelman-data-files/prod/mitelman_db.zip, HTTPSConnectionPool(host='storage.googleapis.com', port=443): Max retries exceeded with url: /mitelman-data-files/prod/mitelman_db.zip (Caused by NameResolutionError("<urllib3.connection.HTTPSConnection object at 0x7fc68479f4d0>: Failed to resolve 'storage.googleapis.com' ([Errno -3] Temporary failure in name resolution)"))[0m [32mDownloading https://compbio.uth.edu/FusionGDB2/tables/FusionGDB2_id.xlsx[0m [31mError downloading https://compbio.uth.edu/FusionGDB2/tables/FusionGDB2_id.xlsx, HTTPSConnectionPool(host='compbio.uth.edu', port=443): Max retries exceeded with url: /FusionGDB2/tables/FusionGDB2_id.xlsx (Caused by NameResolutionError("<urllib3.connection.HTTPSConnection object at 0x7fc6841cdbd0>: Failed to resolve 'compbio.uth.edu' ([Errno -3] Temporary failure in name resolution)"))[0m [1;31m['Mitelman: HTTPSConnectionPool(host=\'storage.googleapis.com\', port=443): Max retries exceeded with url: /mitelman-data-files/prod/mitelman_db.zip (Caused by NameResolutionError("<urllib3.connection.HTTPSConnection object at 0x7fc68479f4d0>: Failed to resolve \'storage.googleapis.com\' ([Errno -3] Temporary failure in name resolution)"))', 'FusionGDB2: HTTPSConnectionPool(host=\'compbio.uth.edu\', port=443): Max retries exceeded with url: /FusionGDB2/tables/FusionGDB2_id.xlsx (Caused by NameResolutionError("<urllib3.connection.HTTPSConnection object at 0x7fc6841cdbd0>: Failed to resolve \'compbio.uth.edu\' ([Errno -3] Temporary failure in name resolution)"))'][0m Traceback (most recent call last): File "/opt/conda/lib/python3.13/site-packages/fusion_report/app.py", line 71, in run Download(params) ~~~~~~~~^^^^^^^^ File "/opt/conda/lib/python3.13/site-packages/fusion_report/download.py", line 22, in init self.download_all(params) ~~~~~~~~~~~~~~~~~^^^^^^^^ File "/opt/conda/lib/python3.13/site-packages/fusion_report/download.py", line 66, in download_all raise _DownloadException_(return_err) fusion_report.common.exceptions.download.DownloadException: ['Mitelman: HTTPSConnectionPool(host=\'storage.googleapis.com\', port=443): Max retries exceeded with url: /mitelman-data-files/prod/mitelman_db.zip (Caused by NameResolutionError("<urllib3.connection.HTTPSConnection object at 0x7fc68479f4d0>: Failed to resolve \'storage.googleapis.com\' ([Errno -3] Temporary failure in name resolution)"))', 'FusionGDB2: HTTPSConnectionPool(host=\'compbio.uth.edu\', port=443): Max retries exceeded with url: /FusionGDB2/tables/FusionGDB2_id.xlsx (Caused by NameResolutionError("<urllib3.connection.HTTPSConnection object at 0x7fc6841cdbd0>: Failed to resolve \'compbio.uth.edu\' ([Errno -3] Temporary failure in name resolution)"))'] The above exception was the direct cause of the following exception: Traceback (most recent call last): File "/opt/conda/bin/fusion_report", line 12, in <module> app.run() ~~~~~~~^^ File "/opt/conda/lib/python3.13/site-packages/fusion_report/app.py", line 78, in run raise _AppException_(ex) from ex fusion_report.common.exceptions.app.AppException: ['Mitelman: HTTPSConnectionPool(host=\'storage.googleapis.com\', port=443): Max retries exceeded with url: /mitelman-data-files/prod/mitelman_db.zip (Caused by NameResolutionError("<urllib3.connection.HTTPSConnection object at 0x7fc68479f4d0>: Failed to resolve \'storage.googleapis.com\' ([Errno -3] Temporary failure in name resolution)"))', 'FusionGDB2: HTTPSConnectionPool(host=\'compbio.uth.edu\', port=443): Max retries exceeded with url: /FusionGDB2/tables/FusionGDB2_id.xlsx (Caused by NameResolutionError("<urllib3.connection.HTTPSConnection object at 0x7fc6841cdbd0>: Failed to resolve \'compbio.uth.edu\' ([Errno -3] Temporary failure in name resolution)"))'] Command error: ['Mitelman: HTTPSConnectionPool(host=\'storage.googleapis.com\', port=443): Max retries exceeded with url: /mitelman-data-files/prod/mitelman_db.zip (Caused by NameResolutionError("<urllib3.connection.HTTPSConnection object at 0x7fc68479f4d0>: Failed to resolve \'storage.googleapis.com\' ([Errno -3] Temporary failure in name resolution)"))', 'FusionGDB2: HTTPSConnectionPool(host=\'compbio.uth.edu\', port=443): Max retries exceeded with url: /FusionGDB2/tables/FusionGDB2_id.xlsx (Caused by NameResolutionError("<urllib3.connection.HTTPSConnection object at 0x7fc6841cdbd0>: Failed to resolve \'compbio.uth.edu\' ([Errno -3] Temporary failure in name resolution)"))'] Work dir: /home/kjozsi/projects/nf-core/rnafusion/work/02/6875646bd857c32d7af5a2e3062eb3 Container: /lustre01/singularity/community-cr-prod.seqera.io-docker-registry-v2-blobs-sha256-ed-ed32f46746a5b33a1b5d597cfe2f62d3b1cfa54638f57cadc5d5158d6a4959d2-data.img

Can you try removing the

fusion_report.log

file from the directory? I think this is the issue since the pipeline checks if the files in the directory are not empty to determine if the reference is correctly created

Thanks a lot. I did that and ran the pipeline several times. Now got some other, seemingly unrelated errors. I'm tackling them now. Based on this error below, I'm rebuilding the starfusion reference. I'll keep you updated. Jan-06 135036.612 [TaskFinalizer-5] ERROR nextflow.processor.TaskProcessor - Error executing process > 'RNAFUSION:STARFUSION_DETECT (UPL25-007280_1106N_25)' Caused by: Process

RNAFUSION:STARFUSION_DETECT (UPL25-007280_1106N_25)

terminated with an error exit status (2) Command executed: STAR-Fusion \ --genome_lib_dir ctat_genome_lib_build_dir \ \ -J UPL25-007280_1106N_25.Chimeric.out.junction \ --CPU 12 \ --output_dir . \ mv star-fusion.fusion_predictions.tsv UPL25-007280_1106N_25.starfusion.fusion_predictions.tsv mv star-fusion.fusion_predictions.abridged.tsv UPL25-007280_1106N_25.starfusion.abridged.tsv if [ -f star-fusion.fusion_predictions.abridged.coding_effect.tsv ]; then mv star-fusion.fusion_predictions.abridged.coding_effect.tsv UPL25-007280_1106N_25.starfusion.abridged.coding_effect.tsv fi cat <<-END_VERSIONS > versions.yml "RNAFUSION:STARFUSION_DETECT": STAR-Fusion: 1.15.1 END_VERSIONS Command exit status: 2 Command output: (empty) Command error: * Error, not able to find file: trans.blast.align_coords.align_coords.dbm * at /opt/conda/lib/STAR-Fusion/util/validate_ctat_genome_lib.pl line 28. ERROR, ctat genome lib: ctat_genome_lib_build_dir does not validate and may require indices to be rebuilt. Visit: https://github.com/STAR-Fusion/STAR-Fusion/wiki/rebuild_ctat_genome_lib_indices for details.

Allright hope that will help 😛