Question: Can I sequence my 10x Genomics libraries with Singular Genomics G4 Sequencing Platform?
Answer: Yes, you can sequence 10x Genomics libraries using the Singular Genomics® G4® Sequencing Platform. 10x Genomics has verified compatibility of our Single Cell 3’ v3.1 Gene Expression libraries with the G4 Sequencing Platform using option 1 below. Please reach out to Singular Genomics regarding testing of other library types.
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. The S1 and S2 adapter sequences are different from the P5 and P7 adapters used in 10x Genomics Illumina-compatible library construction.
Please check Singular Genomics’ table for their recommendations for which workflow to use to adapt 10x libraries to be sequenced on the G4 Sequencing Platform. You have three options to create libraries that are compatible with the Singular Genomics G4 Sequencing Platform, outlined below.
Only Option 1 below has been validated by 10x Genomics as part of the Compatible Partner Program with Singular Genomics. Options 2 and 3 have been tested by Singular Genomics with 10x Genomics libraries but have not been validated by 10x Genomics.
10x Genomics is not able to provide troubleshooting guidance for adapting 10x Genomics libraries for Singular Genomics compatibility, or guidance regarding sequencing configurations, library pooling, custom primer design, or analysis using Singular Genomics workflows. 10x Genomics cannot guarantee success with these downstream workflows nor offer any replacements or goodwill reagents for troubleshooting Singular Genomics workflows. Please reach out to Singular Genomics (care@singulargenomics.com) for assistance with troubleshooting Singular Genomics workflows.
Option 1 - use Singular Genomics primers to add S1 and S2 adapters instead of P5 and P7 adapters during 10x Genomics library preparation.
- 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 by 10x Genomics as part of the Compatible Partner Program with Singular Genomics, for the Chromium Single Cell 3’ v3.1 Dual Index Gene Expression Library preparation protocol only. 10x Genomics will support library preparation up to bead clean up (step 3.4) following adapter ligation.
- 10x Genomics has not validated this approach with other library types. To discuss options for other library types, contact Singular Genomics.
- Resources from Singular Genomics
Option 2: use Singular Genomics primers to add S1 and S2 adapters to existing 10x Genomics libraries.
- Adapt fully constructed existing 10x Genomics libraries using Singular Genomics PCR primers.
- Resources from Singular Genomics
- This approach has not been validated by 10x Genomics.
Option 3: use Singular Genomics Max Read Kit on existing 10x Genomic libraries.
- Use the Max Read (™) Kit on fully constructed existing 10x Genomics libraries. A Max Read Kit generates multiple library fractions of one sample with different sequencing primer binding sites. After combining the fractions, the combined library is sequenced successively with distinct fraction-specific sequencing primers, which enables higher output of short reads without compromising quality.
- Singular Genomics provides two kits for Max Read sequencing, with each kit specific for processing different 10x Genomics library preparations. Compatibility of this kit has been verified by Singular Genomics but has not been tested by 10x Genomics. The table below lists which 10x Genomics libraries are compatible with each Max Read kit. Also refer to the Singular Product Options table.
- This approach has not been validated by 10x Genomics.
Sequencing primers
Singular Genomics uses SP1 and SP2 sequencing primers to prime the sequencing reaction. 10x Genomics library types that use 10x Genomics Dual Index Plate TT already contain binding sites for these sequencing primers. See this article for a list of 10x Genomics library types that use Dual Index Plate TT. These libraries would still require the S1 and S2 adapters to be added to be compatible with the G4 flow cells (see Option 1, 2 and 3 above).
Other 10x Genomics library types prepared with different index plates (e.g. Dual Index Plate NT, TN, NN, TS; see this list), 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. Also refer to Singular’s table for their recommendations for different 10x Genomics library types.
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 Reads per flow cell (1,000 M Reads per run)
- F3: up to 450 M Reads per flow cell (1,800 M Reads per run)
- https://singulargenomics.com/g4/reagents/
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.
Data analysis
FASTQ files output from the Singular demultiplexing software should be compatible with Cell Ranger v7.0.0 or later as long as the standard file naming convention is followed. For questions related to demultiplexing, please reach out to Singular care@singulargenomics.com. For questions related to running Cell Ranger data analysis software, please contact 10x Genomics support@10xgenomics.com.
Resources:
Singular Genomics Application Note: Single Cell RNA Sequencing on the G4
For additional information, please reach directly out to: care@singulargenomics.com
Products: Single Cell Gene Expression, Single Cell Immune Profiling, Single Cell Gene Expression Flex, Single Cell Multiome ATAC+GEX, Visium for Fresh Frozen