cellranger mkref fail/error

Question

[tatsispolychronis]

Hey, I use cellranger mkref to make a custom transcriptome for my invertebrate analysis. SInce i proceeded with 3' OCM, i include both species in the command:

cellranger mkref \
   --genome=DMAN_ref --fasta=/home/***/DMAN1_supertranscripts.fasta --genes=/home/***/DMAN1_supertranscripts_clean.gtf \
   --genome=HLOG_ref --fasta=/home/***/HLOG1_supertranscripts.fasta --genes=/home/***/HLOG1_supertranscripts_clean.gtf \
   --memgb=200 --nthreads=16

and I get this error:

Martian Runtime - v4.0.13
Serving UI at http://apollo:34727?auth=bhYmxHd5fZAd-i3sedOz3DwBd30eJr3htwsDYxfXxb4

Running preflight checks (please wait)...
2025-09-23 12:56:34 [runtime] (ready)           ID.mkref_DMAN_ref_and_HLOG_ref.MAKE_REFERENCE._MAKE_REFERENCE
2025-09-23 12:56:34 [runtime] (run:local)       ID.mkref_DMAN_ref_and_HLOG_ref.MAKE_REFERENCE._MAKE_REFERENCE.fork0.split
2025-09-23 12:56:34 [runtime] (split_complete)  ID.mkref_DMAN_ref_and_HLOG_ref.MAKE_REFERENCE._MAKE_REFERENCE
2025-09-23 12:56:34 [runtime] (run:local)       ID.mkref_DMAN_ref_and_HLOG_ref.MAKE_REFERENCE._MAKE_REFERENCE.fork0.join
2025-09-23 13:02:35 [runtime] (update)          ID.mkref_DMAN_ref_and_HLOG_ref.MAKE_REFERENCE._MAKE_REFERENCE.fork0 join_running
2025-09-23 13:08:36 [runtime] (update)          ID.mkref_DMAN_ref_and_HLOG_ref.MAKE_REFERENCE._MAKE_REFERENCE.fork0 join_running
2025-09-23 13:14:37 [runtime] (update)          ID.mkref_DMAN_ref_and_HLOG_ref.MAKE_REFERENCE._MAKE_REFERENCE.fork0 join_running
2025-09-23 13:16:26 [runtime] (failed)          ID.mkref_DMAN_ref_and_HLOG_ref.MAKE_REFERENCE._MAKE_REFERENCE

[error] mkref has failed: error building reference package
Failed to make genome index with STAR.  This can occasionally be caused by setting the argument `memgb` too low.
Error was from running command '/opt/cellranger-9.0.1/lib/bin/STAR'
Command '['/opt/cellranger-9.0.1/lib/bin/STAR', '--runMode', 'genomeGenerate', '--genomeDir', '/home/***/mkref_DMAN_ref_and_HLOG_ref/MAKE_REFERENCE/_MAKE_REFERENCE/fork0/join-u5885d27ce2/files/reference/star', '--runThreadN', '16', '--genomeFastaFiles', '/home/***/mkref_DMAN_ref_and_HLOG_ref/MAKE_REFERENCE/_MAKE_REFERENCE/fork0/join-u5885d27ce2/files/reference/fasta/genome.fa', '--sjdbGTFfile', '/home/***/mkref_DMAN_ref_and_HLOG_ref/MAKE_REFERENCE/_MAKE_REFERENCE/fork0/join-u5885d27ce2/files/reference/genes/genes.gtf', '--limitGenomeGenerateRAM', '214748364800', '--genomeSAsparseD', '1', '--genomeSAindexNbases', '13', '--genomeChrBinNbits', '9']' returned non-zero exit status 104.

Check stdout and stderr for more information.

I thought it was a STAR issue since by default it asks for 16G of RAM but even thought i asked for 200G it still gives me this error. Gtfs and fasta files have the correct headers. I managed to run mkref for each species individually, and it run properly.

anyone had the same issue or managed to resolve it/has an idea?

Thank you a priori :)

[GenoMax]

You need to provide the genome reference for the --fasta option as noted in https://www.10xgenomics.com/support/software/cell-ranger/latest/analysis/inputs/cr-3p-references#adding-one-or-more-genes-to-your-reference-304f27. Based on the names of your files, it appears that you are using transctiptomes fasta.

[tatsispolychronis]

The thing is that I do not have a genome for these organisms. i made a transcriptome using TRinity and this is what I am feeding it. I do nto think this is the issue tbh, but neither have I seen someone using a transcriptome as a reference.

[GenoMax]

The thing is that I do not have a genome for these organisms.

That is not what cellranger mkref is expecting.

You could consider alternate tools like kallisto bustools or simpleaf. Not sure what is 3'-OCM.