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: NovaSeq X Series instruments are currently supported for sequencing some 10x Genomics libraries. We are currently working to optimize additional 10x Genomics library types on NovaSeq X series instruments. This information is outlined in the article: What considerations are there for sequencing 10x Genomics libraries on NovaSeq X Series instruments?
Update (Oct. 11, 2023): Cell Ranger v7.2.0 and above supports NovaSeq X input run directories for Single Cell Gene Expression and Single Cell Immune Profiling libraries. See release notes here. Please download and install v7.2.0 and above.
Space Ranger v2.1.1 and above supports NovaSeq X input run directories for Spatial Gene Expression and Spatial Gene Expression for FFPE. See release notes here. Please download and install v2.1.1 and above.
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.
If you are unable to update the Cell Ranger or Space Ranger pipeline to a current build (Cell Ranger v7.2 and above or Space Ranger v2.1.1 and above) then you will need to directly run one of Illumina’s demultiplexing pipelines, either bcl2fastq or bcl-convert to generate FASTQ files from NovaSeq X sequencing.
BCL Convert is planned to replace bcl2fastq in the future. If you are generating single cell ATAC or multiome ATAC FASTQ files from NovaSeq X, we recommend running 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: October 2023