cellranger mkref failed

Question

[Dan]

Hi,

I use cellranger mkref to build the customized mm10 genome reference. But it always showes error like this:

cellranger mkref --genome=refdata-cellranger-mm10-3.0.0-custom --fasta=~/Custom/Fas/mm10_combined.fa --genes=~/Custom/gtf/Mus_musculus.GRCm38.93_costom_filtered.gtf
/lila/home/lid/cellranger-3.1.0/cellranger-cs/3.1.0/bin
cellranger mkref (3.1.0)
Copyright (c) 2019 10x Genomics, Inc.  All rights reserved.
-------------------------------------------------------------------------------

Creating new reference folder at /lila/home/lid/refdata-cellranger-mm10-3.0.0-custom
...done

Writing genome FASTA file into reference folder...
...done

Computing hash of genome FASTA file...
...done

Indexing genome FASTA file...
...done

Writing genes GTF file into reference folder...
...done

Computing hash of genes GTF file...
...done

Writing genes index file into reference folder (may take over 10 minutes for a 3Gb genome)...
...done

Writing genome metadata JSON file into reference folder...
...done

Generating STAR genome index (may take over 8 core hours for a 3Gb genome)...
Aug 16 14:30:49 ..... Started STAR run
Aug 16 14:30:49 ... Starting to generate Genome files
Traceback (most recent call last):
  File "/lila/home/lid/cellranger-3.1.0/cellranger-cs/3.1.0/bin/mkref", line 100, in <module>
    main()
  File "/lila/home/lid/cellranger-3.1.0/cellranger-cs/3.1.0/bin/mkref", line 97, in main
    referenceBuilder.build_reference()
  File "/lila/home/lid/cellranger-3.1.0/cellranger-cs/3.1.0/lib/python/cellranger/reference.py", line 243, in build_reference
    mem_gb=self.mem_gb)
  File "/lila/home/lid/cellranger-3.1.0/cellranger-cs/3.1.0/lib/python/cellranger/reference.py", line 486, in index_reference_with_mem_gb
    limit_ram=limit_ram)
  File "/lila/home/lid/cellranger-3.1.0/cellranger-cs/3.1.0/lib/python/cellranger/reference.py", line 507, in index_reference
    tk_subproc.check_call(args)
  File "/lila/home/lid/cellranger-3.1.0/cellranger-cs/3.1.0/tenkit/lib/python/tenkit/log_subprocess.py", line 37, in check_call
    return subprocess.check_call(*args, **kwargs)
  File "/lila/home/lid/cellranger-3.1.0/miniconda-cr-cs/4.3.21-miniconda-cr-cs-c10/lib/python2.7/subprocess.py", line 186, in check_call
    raise CalledProcessError(retcode, cmd)
subprocess.CalledProcessError: Command '['STAR', '--runMode', 'genomeGenerate', '--genomeDir', '/lila/home/lid/refdata-cellranger-mm10-3.0.0-custom/star', '--runThreadN', '1', '--genomeFastaFiles', '/lila/home/lid/refdata-cellranger-mm10-3.0.0-custom/fasta/genome.fa', '--sjdbGTFfile', '/lila/home/lid/refdata-cellranger-mm10-3.0.0-custom/genes/genes.gtf', '--limitGenomeGenerateRAM', '17179869184', '--genomeSAsparseD', '3', '--genomeSAindexNbases', '14', '--genomeChrBinNbits', '18']' returned non-zero exit status -9

Can anybody tell me what is wrong with this?

Thanks Dan

[ATpoint]

How did you customize the gtf, please show the command and an example of the file.

[hongyuchen]

Do you get a method to solve the problem?

[CandiceChuDVM]

I have added 10x genomics' solution below:

[CandiceChuDVM]

I encountered a similar problem. Here is 10x genomics' response:

"It looks like the job failed in the STAR indexing step. This kind of failure is often caused by running out of memory. It could be a transient error or a limitation on the machine.

You can try running it on a node with more available memory (32GB or more). You could also try adding the --memgb option (https://support.10xgenomics.com/single-cell-gene-expression/software/pipelines/latest/advanced/references#mkref) in your command line. You can start with --memgb=32, and if the job fails again, you may need to further increase the value."

[yuri]

I tried use -memgb=64, it doesn't work. I also want to make mm10 genome reference

[GenoMax]

Is there a particular reason you want to make your own? 10x makes mouse genome indexes available for use with cellranger.

[yuri]

Did you get a method to solve the problem? I also met the same problem.

[EricYang]

i met the problem with memgb=300