Question: I have a NovaSeq X run that I’m trying to demultiplex with mkfastq (cellranger, cellranger-arc, spaceranger, cellranger-atac) but keep getting an error, can this be fixed?
Answer: The NovaSeq X is not yet an officially supported sequencing instrument for 10x Genomics products. Below is a list of supported sequencing instruments, applicable for all products: Single Cell Gene Expression, Single Cell Immune Profiling, Single Cell Multiome ATAC + Gene Expression, Single Cell ATAC, Spatial Gene Expression, Spatial Gene Expression for FFPE.
- Illumina® NovaSeq 6000
- Illumina® HiSeq 3000/4000
- Illumina® HiSeq 2500 Rapid Run
- Illumina® NextSeq 500/550
- Illumina® NextSeq 1000/2000
- Illumina® MiSeq
Below is the error produced when a NovaSeq X run directory is used as input to cellranger mkfastq v7.1.0. The mkfastq pipeline bundled with other ranger pipelines (cellranger-arc, spaceranger, cellranger-atac) will display a similar error.
[error] Pipestance failed. Error log at:
GEX_mkfastq_default/MAKE_FASTQS_CS/MAKE_FASTQS/MAKE_FASTQS_PREFLIGHT/fork0/chnk0-u2521874eb1/_errors
Log message:
Traceback (most recent call last):
File "cellranger-7.1.0/external/martian/adapters/python/martian_shell.py", line 648, in _main
stage.main()
File "cellranger-7.1.0/external/martian/adapters/python/martian_shell.py", line 607, in main
self._run(lambda: self._module.main(args, outs))
File "cellranger-7.1.0/external/martian/adapters/python/martian_shell.py", line 564, in _run
cmd()
File "cellranger-7.1.0/external/martian/adapters/python/martian_shell.py", line 607, in
self._run(lambda: self._module.main(args, outs))
File "cellranger-7.1.0/mro/tenkit/stages/make_fastqs/make_fastqs_preflight/__init__.py", line 54, in main
(rta_version, _, bcl_params) = tk_bcl.get_rta_version(args.run_path)
File "cellranger-7.1.0/lib/python/tenkit/bcl.py", line 140, in get_rta_version
application_version = tree.getroot().find("ApplicationVersion").text
AttributeError: 'NoneType' object has no attribute 'text'
The reason for this error is because the format of the RunParameters.xml file has changed in NovaSeq X run directories and this new format is not yet compatible with the mkfastq pipeline.
To generate FASTQ files from NovaSeq X, you will need to directly run one of Illumina’s demultiplexing pipelines, either bcl2fastq or bcl-convert. The following are links by product to our support pages with instructions, appropriate settings, and example sample sheets to be used for direct demultiplexing with Illumina’s software:
- 3’ Gene Expression
- 5’ Immune Profiling
- Single Cell Multiome ATAC + Gene Expression
- Single Cell ATAC
- Spatial Gene Expression
- Spatial Gene Expression for FFPE
The FASTQ files that are output by mkfastq, bcl-convert, and bcl2fastq can successfully be used as input to the cellranger, cellranger-arc, spaceranger, or cellranger-atac pipelines applicable for your assay.
Related KB article: Is mkfastq really needed to demultiplex, or can we use bcl2fastq or bcl-convert?
Products: All
Last updated: June 2023