Question: Can I sequence my libraries with Singular Genomics?
Answer: Yes, you can sequence 10x Genomics libraries using the Singular Genomics G4™ Sequencing Platform.
The G4 Sequencing Platform by Singular Genomics™ requires S1 and S2 adapter sequences to be added to the ends of each library fragment for clustering on flow cells. These are distinct from the P5 and P7 adapters used in our Illumina compatible library construction.
You have two options to create libraries that are compatible with the Singular Genomics G4 Sequencing Platform:
- Use Singular Genomics Dual Indexed Primers (SG t-UDI - Set of 24 [Set A], catalog # 700143) or Non-indexed primers (G4 Non-Indexed Library Prep t-Primers (24 Rxns), catalog # 700144) during library preparation instead of Illumina primers during the sample index PCR step. This approach has been validated for Chromium 3’ Gene Expression Library preparation protocol. Singular Genomics is an accredited member of the Compatible Partner Program. 10x Genomics will support library preparation up to bead clean up (step 3.4) following adapter ligation. For other libraries please contact Singular Genomics support.
- Adapt fully constructed existing 10X Genomics libraries using Singular Genomics PCR primers. 10x Genomics cannot guarantee success with unsupported downstream workflows and we are unable to offer any replacements or goodwill reagents due to unsatisfactory results from Singular Genomics workflows.
- Guidelines to adapt libraries and add indexes: https://singulargenomics.com/wp-content/uploads/2023/06/Adapting-Library-Insert-600024.pdf
- Guidelines to adapt libraries that are already indexed: https://singulargenomics.com/wp-content/uploads/2023/06/Adapting-Library-Retain-Index-600025.pdf
Singular Genomics uses SP1 and SP2 sequencing primers to prime the sequencing reaction. The following 10x assays already contain binding sites for these sequencing primers:
- Chromium Single Cell Gene Expression (i.e. 3’ v3.1)
- Gene Expression libraries only
- Spatial Gene Expression for Fresh Frozen (Visium for Fresh Frozen)
- Chromium Single Cell Immune Profiling (i.e. 5’ v2)
- Gene Expression, V(D)J, and 5’ CRISPR libraries
- Single Cell Multiome ATAC + Gene Expression
- Gene Expression libraries only
These libraries do still require the S1 and S2 adapters to be added to be compatible with the G4 flow cells. Protocols for incorporating these adapters can be found on the Singular Genomics website.
Other 10x assays may require custom primers for converting the library for compatibility and for sequencing. Please reach out to Singular Genomics for assistance in designing custom primers.
Do samples always require indexing for sequencing on the Singular Genomics G4 Sequencing Platform?
With the G4 Sequencing Platform, users have the option to load libraries on different lanes of a flow cell and on separate flow cells. There are 4 fluidically independent lanes per flow cell. A sequencing run can be comprised of 1, 2, 3 or 4 flow cells.
There are two types of flow cells:
F2: up to 250 M Read Pairs per flow cell (1,000 M Read Pairs per run)
F3: up to 450 M Read Pairs per flow cell (1,800 M Read Pairs per run)
Therefore depending on the application and the number of reads required per sample; it is possible to physically separate samples across lanes or flow cells rather than having to index and pool and demultiplex samples.
Singular Genomics Application Note: Single Cell RNA Sequencing on the G4
For additional information, please reach directly out to: firstname.lastname@example.org
Products: Single Cell Gene Expression, Single Cell Immune Profiling, Single Cell Multiome ATAC+GEX, Spatial Gene Expression for Fresh Frozen