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San Diego-based Singular Genomics was founded in 2016 with the goal of overcoming the limitations of the available sequencing tools. Using feedback from scientists and clinicians, Singular designed and launched their G4ä Sequencing System. It stands out as a powerful benchtop sequencing platform with significantly reduced run times, delivering high-quality data in less than 24 hours across a wide array of applications. Notably, the G4 is capable of sequencing up to four human genomes in a single day, producing 2.5 times more data per day than the leading benchtop alternative.
G4 Sequencing Platform
Key attributes of Singular's G4 include its unique flow cell design, the run scalability, and the flexibility and customization of its sequencing runs. Darius Fugere, VP of Marketing at Singular Genomics, emphasized that the G4 can generate daily results across 1‒4 flow cells and 8 different run sizes, which eliminates batching bottlenecks and enables on-demand, cost-effective sequencing.
The flow cell design, Fugere noted, is particularly significant because it features 4 independently addressable lanes, allowing for 4‒16 lanes per run. This flexibility mitigates the need for sample pooling and streamlines project management. It is particularly advantageous for labs handling variable volume and time-sensitive samples as it ensures efficient use of resources and swift turnaround times.
The G4 system is also marked by its accuracy, offering sequencing data with 80‒90% of bases ≥ Q30, metrics that are on par with leading benchtop instruments. Fugere detailed that the G4 has enhanced speed and a high-resolution optical system that works seamlessly with novel Rapid sequencing by synthesis (SBS) chemistry and specially designed microfluidics to achieve sub-3-minute cycle times. In addition, the proprietary 4-color rapid SBS chemistry uses innovative nucleotides, specially engineered enzymes, and advanced molecular biology methods to deliver accurate paired-read sequencing at increased speed.
Fugere also highlighted that the G4 demonstrates impressive plug-and-play capabilities. It is designed to integrate with top library prep kits with 18 validated partners and comes with Singular's custom adapters and indices, fitting seamlessly into many existing sequencing workflows. Furthermore, it easily connects with leading cloud storage providers and generates easily integrable, demultiplexed FASTQ files for current bioinformatic pipelines.
Sequencing process
“Sequencing on the G4 is done by rapid 4-color sequencing-by-synthesis that was developed entirely in-house from the ground up,” Fugere explained. During the sequencing process, a cycle begins with the incorporation of fluorescently labeled nucleotides, which have a reversible terminator, into a growing strand of DNA. The cycle ends with the cleavage of the terminator allowing the next cycle to advance. Each base is connected to a uniquely colored fluorophore that is detected by the high-speed and high-resolution imaging system of the G4. “With this novel chemistry and rapid fluidic and imaging systems, the G4 is able to complete a cycle of sequencing in under 3 minutes and process over a billion pixels per second,” added Fugere.
Common applications
One application well suited for the G4 Sequencing Platform is RNA sequencing. Representative gene expression data presented by Singular shows a comparable performance against other leading sequencers. Additionally, the G4 system is particularly well adept for single-cell RNA sequencing and compatible with existing upstream library preparation kits. Singular Genomics has continued to expand their single-cell capabilities through partnerships with 10x Genomics, Parse Biosciences, and Takara Bio.
The G4 system has also shown strong results for whole exome (WES) and whole genome sequencing (WGS) applications. WES data from the G4 has displayed high accuracy for both SNP and indel calling using the standard HG001 reference. This either met or exceeded the performance of DeepVariant models produced with other sequencing platforms. For WGS, users can obtain 30x coverage of four human genomes in less than 24 hours with a single G4 sequencing run containing four F3 flow cells run in parallel. In addition, sequencing data from WGS experiments run with reference samples on the G4 Platform produced efficient and accurate data that maintained uniform coverage of the high-confidence regions.
“The G4 is a highly versatile system,” stated Fugere. This adaptability is most evident in its adoption in academic core laboratories, which routinely undertake a variety of sequencing projects.
“Three of our five initial shipments were to academic core labs, including Harvard's Beth Israel Deaconess Medical Center, the Medical College of Wisconsin, and the University of New Mexico,” explained Fugere. “At Harvard, the flexibility and speed of the G4 have been key in allowing them to utilize a single instrument to run single-cell and spatial experiments with varying sample volumes and output requirements. At the Medical College of Wisconsin, the team has been impressed with the high quality of the sequencing data and single-day turnaround times. And at the University of New Mexico, the flexibility of the G4 is ideally suited to address their diversity of applications.”
Advances and the future
Singular Genomics has been making continuous improvements in flow cell throughput. The throughput range of the F2 and F3 flow cells has been increased to up to 250 million and up to 450 million reads, respectively. This means that up to 1.8 billion reads per run can be achieved using F3 flow cells for certain applications. They've also enhanced the quality specifications for the G4 Sequencing Platform, reaching 80‒90% bases ≥ Q30.
Recent advances also include the company’s introduction of their Max Readä Kits, designed to further increase the output of the G4 benchtop system for single-cell sequencing to 3.2 billion reads per run. These kits, tailored to be compatible with popular 10x Genomics assays, allow users to perform their routine single-cell projects with increased flexibility. With an expected cost of $1 per million reads, they also provide 800 million reads per flow cell, at a high quality (80‒90% bases ≥ Q30).
Singular Genomics is also currently developing PX, the first integrated single-cell sequencing and spatial profiling platform. “The PX system leverages Singular’s proprietary sequencing technology, applying it as an in situ readout to look at RNA and proteins in single cells and tissue,” explained Fugere. “The platform is still in development and we have kicked off work with early Technology Access Partners.” Although specifics regarding the PX system remain undisclosed, anticipation is high within the scientific community as they eagerly look forward to the unveiling of this groundbreaking technology.