The genomics world is buzzing. A few weeks ago, PacBio introduced SPRQ chemistry for its Revio system, a leap forward for HiFi sequencing technology that’s igniting excitement among researchers worldwide. Promising groundbreaking efficiency and affordability at the same exceptional accuracy and read length, this chemistry marks a pivotal moment for those tackling complex genomes and challenging research questions. SPRQ chemistry isn’t just an upgrade; it’s a game-changer for tackling some of genomics’ toughest challenges.
Join us as we dive into the details of SPRQ chemistry, how it compares to the first iteration of Revio technology, and what it means for the future of genomics.
What is SPRQ chemistry?
SPRQ (pronounced “spark”) chemistry is enabled by a new sequencing polymerase and optimized reagents that ensure more DNA molecules reach the surface of a SMRT Cell during the loading process. This optimization kickstarts improved efficiency throughout the entire sequencing workflow. With increased output, coupled with reduced time, DNA input, and cost, SPRQ chemistry changes the game for HiFi sequencing.
Low input, high impact
This revolutionary chemistry improvement transforms the efficiency of HiFi sequencing and opens up new avenues for groundbreaking research – and it all starts with the sample input. With more efficient SMRT Cell loading, SPRQ chemistry on the Revio system now requires only 500 ng of DNA into library prep. The library prep protocols remain the same, but because less DNA is needed to load a SMRT Cell, less can be used for library prep. Even better, the 500 ng is the total input across samples. This means that if you were to multiplex two samples on a SMRT Cell, you could start with 250 ng each into library prep.
This dramatic reduction in input unlocks entirely new opportunities for genomics, allowing HiFi sequencing for more precious sample types, overcoming challenges that have traditionally limited the feasibility of long-read sequencing projects.
Neonatal research, for example, often relies on a few drops of blood from a heel prick. This small sample volume that had previously rendered sequencing more difficult and less reliable now becomes more accessible, opening up the opportunity for earlier, more comprehensive insights into genetic conditions and the potential to inform research in life-changing interventions in pediatric care.
Reducing DNA input with SPRQ chemistry also has the potential to impact sequencing projects of very large scale, like population genetics studies that rely on saliva as the sample source. Because it is readily available with easy and non-invasive collection, saliva has become an attractive widescale alternative to blood samples. Unlike blood however, DNA yield is typically lower in saliva. These challenges require high-quality sequencing that is less sensitive to input amount to achieve greater precision in variant detection and mapping. The PacBio protocol for extracting DNA from saliva using Nanobind kits along with the increased sample input efficiency of SPRQ chemistry empowers researchers to channel the power of population genomics studies and work toward actionable population health initiatives.
Illuminating multiomic insights
A paired update to Revio instrument software also unlocks a new type of on-board methylation detection, with the inclusion of 6mA calling and 10% increased accuracy for 5mC CpG detection. The 6mA calling simplifies a key step of the Fiber-seq method1, which overlays chromatin architecture with 5mC methylation, providing a deeper view of the epigenome and harnessing the power of the Revio system for multiomic sequencing. With Fiber-seq and the epigenetic enhancement coming from SPRQ chemistry, researchers can simultaneously generate four high-quality haplotype-resolved ‘omes – the genome, CpG methylome, chromatin epigenome, and transcriptome – from a single Revio run.
The promise of SPRQ chemistry to expand the impact of multiomics is acclaimed by fibertools developer Mitchell Vollger:
“I am excited to see an on-instrument 6mA caller for the Revio system with sufficient accuracy for use in Fiber-seq and validated against our 6mA caller (fibertools). This will help simplify the computational Fiber-seq protocol, significantly reduce the storage requirements, support further adoption, and provide important computational support for future chemistries.”
– Mitchell Vollger, PhD, University of Washington
The improvements in 5mC accuracy plus the addition of 6mA calling has the potential to revolutionize our understanding of critical biological systems, like how regulatory processes contribute to cancer evolution. Recent work presented at the American Association for Cancer Research conference showed how tumor-specific hypermethylation in the promoter of tumor suppressor genes can act as a second hit in samples with unidentified driver mutations when sequenced with SBS short reads. On-board 6mA calling for Fiber-seq adds an additional layer of information in this quest to understand how regulatory changes impact cancer development.
Reimagining the potential of the human genome
This transformation has researchers redefining the possibility of the human genome, as HiFi sequencing technology maintains the same exceptional performance at different coverage depths than used for other technologies.
Choosing the right level of sequencing coverage is a critical first step for project planning and requires a careful balance between the complexity of the genome and the targeted variants, the desired sensitivity, and available resources. For certain research projects, it is beneficial to strive for as much coverage as possible, but for many scenarios, increasing coverage does not always yield unbounded increases in performance. This prompts the question: what coverage level is right for my application?
With equivalent industry-leading variant calling performance between SPRQ and the Revio original chemistry, HiFi sequencing demonstrates excellent small variant (SNV and indel) calling at 20x coverage, with highly accurate structural variant detection with as little as 10x coverage.
In a study led by researchers at Radboud University Medical Center2 examining clinically relevant germline variants identified at 30x coverage, a 20x HiFi genome is shown to be sufficient to call 96.2% of these variants, with a 15x HiFi genome capturing 89%. In a conversation with GenomeWeb, principal investigator Alexander Hoischen acknowledges the 20x HiFi genome as “already extremely good” and discusses the potential for his team to sequence 20x genomes in working practice going forward.
Groundbreaking efficiency and affordability
These chemistry improvements with SPRQ translate to groundbreaking efficiency and affordability for HiFi sequencing on the Revio across applications. At a 33% increase from the initial Revio technology, each SMRT Cell yields 120 Gb, equivalent to two 20x human genomes per SMRT Cell. With the same Revio run times, this boost in yield translates to faster projects over time. This generates 480 Gb per run and 2,500 human genomes at 20x coverage per year at a cost of ~$500/genome ($750/genome at 30x coverage).
This isn’t just faster, cheaper sequencing – SPRQ represents the gateway to discovering more. This increased output, coupled with reduced time, DNA input, and cost, allows researchers to tackle larger and more complex projects than ever before. The capability of the Revio system to sequence four SMRT Cells independently generates the power of sequencing multiple sample types and different dimensions of the genome in the same run. For example, a tumor/normal genome and full-length or single-cell transcriptome can be sequenced together, enabling a more comprehensive view of multiomic landscapes simultaneously and accelerated advancements in research.
This revolutionary combination of efficiency, affordability, and versatility is redefining what researchers can achieve, fast-tracking the pace of discovery across research in rare and inherited disease, cancer, and population and ecosystem health.
Ready to ignite your research?
Explore the Revio product page and download SPRQ datasets.
SPRQ consumables are already available for order with shipping to begin in December 2024. New to Revio? Connect with a PacBio scientist for more information.
References
- Stergachis, A. B., Debo, B. M., Haugen, E., Churchman, L. S., & Stamatoyannopoulos, J. A. (2020). Single-molecule regulatory architectures captured by chromatin fiber sequencing. Science, 368(6498), 1449-1454.
- Hoeps, W., et al. (2024). HiFi long-read genomes for difficult-to-detect clinically relevant variants. medRxiv, 2024-09.