Transform Your Single Cell RNA Sequencing with G4
April 18, 2023
Changes in cellular gene expression can have profound impacts on biological systems; these changes are only now starting to be understood, largely through single cell RNA sequencing (scRNA-Seq). While the advancements in genomics, transcriptomics, immunology, developmental biology, and cancer research are evolving rapidly, the impact of scRNA-Seq has been limited by complex sample preparation, workflows requiring sample pooling, long turnaround times, and high costs. The G4 Sequencing Platform is designed to deliver fast, flexible, and cost-efficient sequencing to empower applications like scRNA-Seq that will strengthen our understanding of cellular diversity.
G4: Uniquely Suited to Power scRNA-Seq Applications
G4 has been intentionally engineered to address the limitations in leading benchtop sequencing platforms and deliver cost-efficient sequencing.
Speed and Accuracy
G4 delivers highly accurate and reproducible data with rapid, single-day turnaround times. A novel 4-color rapid sequencing by synthesis (SBS) chemistry and a proprietary imaging technology work in concert to shorten sequencing cycle times and reduce run times.
Flexibility
G4 offers a unique flow cell design with customizable run configurations and the ability to manage projects by run, flow cell, or lane for unmatched operational flexibility. Each sequencing run can utilize 1–4 independent flow cells with each flow cell accommodating up to 4 single cell samples. Users can sequence only 1 sample in a run, or sequence up to 16 different single cell samples and separate them into individual lanes to reduce barcode incompatibility and error.
Cost-Efficiency
The two different read densities of the F2 and F3 flow cells offer users the option to select a flow cell that matches sample throughput, eliminating the requirement of pooling samples to fill a large flow cell. By reducing reagent waste and unused flow cell capacity, sequencing becomes more cost-effective.
| F2 Flow Cell | F3 Flow Cell | Max Read2 | |
|---|---|---|---|
| Paired Reads Delivered / Flow Cell | 200M | 400M | 800M |
| Paired Reads Delivered / Run | 800M | 1,600M | 3,200M |
| # Samples / Flow Cell1 | 1 | 2 | 4 |
| # Samples / Run1 | 4 | 8 | 16 |
| Price / M Paired Read | $3.0 | $1.8 | $1.0 |
G4 can process up to 4 flow cells in parallel. Number of reads and sequencing output shown is per flow cell.
¹Assumes 28×91 read configuration, 10,000 cells/sample and 20,000 paired reads/cell
²Currently only compatible with 10x Genomics Chromium™ 3′ and 5′ Gene Expression assays and Visium™ Spatial Gene Expression. Kits allow for 1 sample per lane and cannot be run at the same time as F2 or F3 flow cells.
Workflow Compatibility
G4 easily integrates into existing lab ecosystems offering plug-and-play compatibility with common scRNA-Seq library preparation workflows from 10x Genomics, Parse Biosciences, and Takara Bio (Figure 1). Data generated is in the form of demultiplexed FASTQ files that integrate seamlessly with existing bioinformatics pipelines.
Figure 1. 10x Genomics® Chromium single cell workflow using the G4 Sequencing Platform.
Max Read Kit for Single Cell Sequencing
The Max Read Sequencing Kit maximizes throughput and decreases costs for single cell sequencing applications. With 4 single cell samples run in 1 flow cell, the Max Read kit delivers 800 million reads. With the scalable flow cell capacity of the G4, 4 flow cells can sequence 16 samples and yield 3.2 billion total reads per run, at a cost of only $1 per million reads.
The Max Read Kit is designed to complement the format of popular 10x Genomics workflows and delivers highly accurate data with single-day turnaround on the G4.
Comparative Study Between Performance of the G4 and the Illumina NextSeq® 2000
In a study conducted to evaluate the performance of the G4 Sequencing Platform compared to the Illumina NextSeq 2000, 10x Genomics Chromium Single Cell 3’ expression libraries were prepared from 7,000 donor PBMCs using the 10x Chromium Next GEM Single Cell 3’ Reagent Kits v3.1 (Dual Index) protocol. Prepared cDNA was split into 2 samples and PCR-amplified using either the Singular Genomics non-indexed PCR primers for sequencing on the G4 Platform or the 10x Genomics Dual Index TT Set A for sequencing on the Illumina NextSeq 2000. FASTQs were processed using the same bioinformatics pipeline.
Data analysis demonstrated high reproducibility of single cell gene expression profiles between technical replicates on the G4 (R2=0.9949) (Figure 2A, 2B).
Figure 2(A) UMAP embedding of single cell gene expression profiles obtained from technical replicate sequencing of a PBMC library.
(B) Spearman’s correlation of average gene expression across technical replicates.
Data also indicated highly comparable single cell gene expression profiles generated on G4 and NextSeq 2000 (R2=0.9819). (Figure 3A, 3B).
Figure 3(A) UMAP embedding of a single gene expression profiles obtained from sequencing of a PBMC library on the G4 and NextSeq 2000 platforms
(B) Spearman’s correlation of average gene expression across platforms, calculated as the library-size normalized, log-transformed UMI counts per gene
The G4 Sequencing Platform demonstrates excellent technical accuracy and reproducibility for single cell analysis via the 10x Chromium Next GEM Single Cell 3’ Reagent Kits v3.1 (Dual Index). In light of these results, Singular Genomics is recognized as a member of the Compatible Partner Program (CPP) for 10x Genomics. Additional comparative data on single cell gene expression profiles and cell type clustering can be found in an application note on our single cell RNA sequencing webpage.
Single cell sequencing will reveal the true scope of cellular diversity, powered by technologies that provide faster, more cost-efficient sequencing. G4 seamlessly integrates into existing scRNA-Seq workflows and laboratories can benefit from unique flow cell flexibility that enables users to match flow cell capacity more closely to sample throughput demand. Flow cell flexibility combined with shorter cycle times enables faster turnaround times, reduces reagent waste, and results in more cost-effective sequencing.
Learn more about scRNA-Seq with G4 by downloading our application note.
Contact us to request a quote or schedule time with a member of the Singular Genomics team to learn more about the unique capabilities of the G4 Sequencing Platform.
References
- G4® Single Cell RNA-Sequencing Performance with the 10x Genomics® Chromium Single Cell 3’ Gene Expression Kit. Singular Genomics Systems, Inc. (September 2023).
