Question: What considerations are there for sequencing 10x Genomics libraries on NovaSeq X Series instruments?
Answer: The compatibility of NovaSeq X Series instruments has been validated by 10x Genomics for the library types in the table below. The table lists the loading concentration and % PhiX input used for 10x Genomics testing, as well as the observed % PhiX alignment and % clusters passing filter (PF) for a typical run.
NovaSeq X Plus loading guidance and expected outcome for run metrics
| Product | Library Type | Sequencing Configuration (R1, i7, i5, R2) | Library loading concentration | %PhiX input | %PhiX aligned | Cluster %PF |
| Chromium Single Cell 3’v4 Dual Index (GEM-X) | Gene Expression | 28, 10, 10, 90 | 150-200pM | 1% | 0.5-2% | ~70% |
| FB - Cell Surface Protein | ||||||
| Chromium Single Cell 3' v3.1, 3' HT Dual index (Next GEM) | Gene Expression | 28, 10, 10, 90 | 150-200pM | 1% | 0.5-2% | ~70% |
| FB - Cell Surface Protein | ||||||
| FB - CRISPR | ||||||
| FB - CellPlex | ||||||
| Chromium Single Cell 5’v3 Dual Index (GEM-X) | Gene Expression | 28, 10, 10, 90 | 150-200pM | 1% | 0.5-2% | ~70% |
| V(D)J | ||||||
| FB - Cell Surface Protein | ||||||
| FB - CRISPR | ||||||
| ChromiumSingle Cell 5' v2, 5' HT Dual index (Next GEM) | Gene Expression | 26, 10, 10, 90 | 150-200pM | 1% | 0.5-2% | ~70% |
| V(D)J | ||||||
| FB - Cell Surface Protein | ||||||
| FB - CRISPR | ||||||
| Chromium Single Cell Gene Expression Flex, singleplex | Gene Expression | 28, 10, 10, 90 | 150-200pM | 1% | 0.5-2% | ~70% |
| FB - Cell Surface Protein | ||||||
| Chromium Single Cell Gene Expression Flex, multiplex | Gene Expression | 28, 10, 10, 90 | 150-200pM | 10% | 5-10% | ~70% |
| FB - Cell Surface Protein | ||||||
| Chromium Single Cell Multiome ATAC + Gene Expression | Multiome Gene Expression | 28, 10, 10, 90 | 150-200pM | 1% | 0.5-2% | ~70% |
| Multiome ATAC | 50, 8, 24, 49 | 100-150pM | ||||
| Chromium Single Cell ATAC v2 | ATAC | 50, 8, 16, 49 | 100-150pM | 1% | 0.5-2% | ~70% |
| Visium CytAssist Spatial Gene Expression (V2) | Gene Expression | 28, 10, 10, 50 | 300-400pM | 1% | 3-8% | ~70% |
| Protein | ||||||
| Visium HD Spatial Gene Expression | Gene Expression | 43, 10, 10, 50 | 300-400pM | 1% | 3-8% | ~70% |
| Spatial Gene Expression for Fresh Frozen | Gene Expression | 28, 10, 10, 90 | 150-200pM | 1% | 0.5-2% | ~70% |
PhiX
Given the tendency of some 10x Genomics libraries to show over- or under-representation of PhiX on the NovaSeq X Plus instrument, we have indicated the range of % PhiX alignment typically reported from sequencing runs using the loading conditions and % PhiX inputs listed in the table above. We recommend the above loading concentrations and % PhiX inputs for the libraries listed. The % PhiX aligned and Cluster %PF may vary between runs/instruments.
For Visium CytAssist Spatial Gene Expression and Visium HD Spatial Gene Expression libraries, it may be possible to use less than 1% PhiX input. Limited testing was performed with 0.5% PhiX input, which resulted in ~1%-3% PhiX aligned. When pipetting lower volumes to achieve 0.5% PhiX input, pipetting accuracy is critical.
Library pooling
We have tested and recommend pooling Feature Barcode libraries with their corresponding Gene Expression libraries for sequencing, to increase nucleotide diversity.
It may be feasible to pool libraries from different 10x Genomics products together, if they share the same sequencing configurations and the same recommended loading concentrations. For example, we have successfully pooled GEM-X Single Cell 3’ and 5’ Gene Expression libraries together on NovaSeq X Series Instruments. We have not extensively tested pooling other combinations of 10x Genomics library types on NovaSeq X Series instruments.
We have not tested and do not recommend pooling libraries with different sequencing configurations. (eg. pooling Visium HD libraries with other 10x Genomics library types).
Loading concentrations
The library loading concentrations in the table above were based on library quantification by qPCR. Alternative quantification methods such as Qubit or Bioanalyzer may report different concentrations than KAPA qPCR due to inherent differences in the technologies. See: How do alternative quantification methods other than qPCR impact library loading?. For assistance assessing over-or underloading of the sequencer, please reach out to techsupport@illumina.com.
Index color balance
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 Series instruments, Illumina recommends avoiding index combinations which only have signals from A bases, G bases, or G+A bases in any given cycle. Refer to Illumina’s guidance on index color balancing and this article:
Demultiplexing
For information regarding whether to select forward or reverse complement orientation for the i5 index, please see the following article from Illumina: Guidelines for reverse complementing i5 sequences for demultiplexing.
To generate FASTQ files from NovaSeq X Series instruments, you will need to directly run Illumina’s demultiplexing pipeline, BCL Convert is recommended. Please see the following articles for more details:
- Does mkfastq work with NovaSeq X data?
- How do I generate single cell ATAC or multiome ATAC FASTQ files from NextSeq or NovaSeq X?
- How to setup a 10x Genomics sample sheet in Illumina's BaseSpace for demultiplexing
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 Spatial Gene Expression, Visium HD