Question: What considerations are there for sequencing 10x Genomics libraries on NovaSeq X?
Answer: 10x Genomics libraries are compatible with sequencing on the NovaSeq X; however, 10x Genomics has not tested running our libraries on NovaSeq X and therefore it is not an officially supported sequencer at this time. Thus, we do not have official recommendations for loading concentrations or pooling considerations. 10x Genomics and Illumina are working together to perform validation testing. 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
For assistance assessing over-or underloading of the sequencer, please reach out to firstname.lastname@example.org.
To generate FASTQ files from NovaSeq X, you will need to directly run one of Illumina’s demultiplexing pipelines, either bcl2fastq or bcl-convert. Please see the following article for more details: Does mkfastq work with NovaSeq X data?
For information regarding which i5 index orientation to use, please see the following article from Illumina: Guidelines for reverse complementing i5 sequences for demultiplexing
Please note that – as for all sequencing systems – color balance in the index read is important for optimal quality and demultiplexing results. When pooling samples on the NovaSeq X, Illumina recommends avoiding index combinations which only have signal from G bases or G+A bases in any given cycle.
Different fragment sizes may cluster preferentially on NovaSeq X. As such, pooling libraries with different fragment lengths may result in bias towards certain fragment sizes. The pooling ratios recommended by 10x Genomics for other Illumina sequencers may not reflect what is needed for pooling on NovaSeq X and may require optimization.
Illumina has published run data for sequencing runs on NovaSeq X using our Multiome ATAC + Gene Expression assay. You can learn more by reading the following blog post:
NovaSeq X data generated by Illumina with 10x Multiome libraries are also highlighted in this application note: High-accuracy next-generation sequencing with the NovaSeq™ X Series
Products: Single Cell Gene Expression, Single Cell Immune Profiling, Single Cell Gene Expression Flex, Single Cell Multiome ATAC + Gene Expression, Single Cell ATAC, Visium for Fresh Frozen, Visium for FFPE, Visium CytAssist Spatial Gene and Protein, Visium CytAssist for FFPE