Long-read whole genome sequencing, available for years, has driven numerous advancements, including fewer gaps in genome assemblies, more accurate assessment of gene duplications, clearer determination of gene network interactions across chromosomes, and the correction of numerous errors in previous genome assemblies based only on short-read sequencing [1].

PacBio’s high fidelity (HiFi) platforms hold particular value due to their generation of long reads (around 10 kb) with very high accuracy. However, the significant cost compared to other long-read sequencing platforms limited accessibility for many researchers. This barrier has been overcome by the PacBio Revio, the latest HiFi platform that delivers significantly more data at a lower, more affordable price point.

What is the PacBio Revio and how does it compare?

The Revio is the latest platform from PacBio Biosciences and represents a significant upgrade from the previous Sequel II. Comparatively, the Revio offers a 15-fold increase in HiFi read output, with 90% of bases ≥Q30 and a median read accuracy ≥Q30. These HiFi reads typically span 15 to 18 kb. Additionally, the Revio requires 50% fewer consumables and boasts a simpler and more cost-effective workflow compared to the Sequel II [2]. 

Although the Revio is more expensive at $779,000 [3], while the Sequel II is $495,000 [4], the price savings come from the lower reagent costs and  higher Revio output. For example, one of our top PacBio partner facilities charges ~$2,045.00 for Revio library prep and sequencing for an expected output of 90 Gb, roughly equivalent to sequencing a human genome at 30X coverage. This same facility charges ~$2,895.50 for Sequel II library prep and sequencing for an expected output of 30 Gb. In order to get the same output as 1 Revio SMRT cell, 3 Sequel II SMRT cells would be needed, so the actual cost for 90 Gb would be closer to $8,686.50. 

However, there are potential downsides to consider when choosing the Revio over the Sequel II. The Revio demands a higher amount of high-quality, high molecular weight (HMW) DNA, requiring at least 3 μg for whole genome sequencing, while the Sequel II excels in low-input studies, even generating data with as little as 5 ng of DNA using its ultra low-input workflow [5]. Furthermore, the Revio requires library molecules longer than 3 kb. Therefore shorter amplicons, Iso-Seq (RNA) libraries, and 10X Genomics single-cell RNA-Seq libraries necessitate a specific MAS-seq protocol with dedicated kits that concatenate these shorter molecules for compatibility with the Revio [6].

Overall, when compared to other PacBio long-read instruments, the Revio represents a clear advancement in nearly every aspect, provided sufficient high-quality input material is available. This upgrade translates to substantial cost savings and accessibility for researchers who previously lacked the resources for HiFi long-read sequencing.

When should the PacBio Revio be considered?

PacBio Revio sequencing should be considered if you want to:

• Detect complex structural variants, such as large inversions, deletions, or translocations
• Accurately map highly repetitive genome regions
• Polish short-read de novo assembly genomes
• Perform full-length amplicon sequencing, like 16S
• Directly detect methylation [7]

How can Genohub help?

At Genohub, we know that each research next-generation sequencing project is unique, so we will take the time to understand your specific project goals and help define precise project specifications. There is no charge for you for this initial consultation or any of our services. Once we have a set of well-defined project specifications we can get you quick and accurate quotes from our NGS partners around the globe, compare all the different quotes for you if needed, and then connect you directly with our partner with the best quote, all using our easy-to-use online platform  We review all quotes to ensure that they meet your project needs and that they specify measurable quality guarantees. If and when you decide to move forward with the project, we will actively supervise and manage it to make sure that all quality and turnaround guarantees are met.

Genohub’s PacBio sequencing partners are experts in every step of the PacBio Revio long-read sequencing process, including high-quality high-molecular weight (HMW) DNA extraction, library preparation, sequencing, and data analysis.

Our partnering providers can recommend the appropriate PacBio platform and sequencing depth based on your specific project goals and sample amount. They also have experience extracting from many different types of biological samples, and have optimized protocols to obtain the highest quality HMW DNA.

We know that each research project is unique, so we have partners who are also open to working with your custom samples and analysis needs! Get started today by letting us know about your long-reads sequencing project here: https://genohub.com/ngs/ .

References

1. Marx, V. Method of the year: long-read sequencing. Nat Methods 20, 6–11 (2023). https://doi.org/10.1038/s41592-022-01730-w
2. PacBio. (2024). REVIO SYSTEM: Reveal more with accurate long-read sequencing at scale. https://www.pacb.com/revio/
3. Pacific Biosciences of California, Inc. (2023, January 9). PacBio Announces Record Order, Including Orders for 76 Revio Systems Received in the Fourth Quarter of 2022. https://www.pacb.com/press_releases/pacbio-announces-record-orders-including-orders-for-76-revio-systems-received-in-the-fourth-quarter-of-2022/#:~:text=The%20Revio%20System%20has%20a,for%20the%20300%2Dcycle%20kit. 
4. Han, AP. (2020, October 8). Pacific Biosciences’ New Sequel IIe System Puts Focus on High Read Accuracy. https://www.genomeweb.com/sequencing/pacific-biosciences-new-sequel-iie-system-puts-focus-high-read-accuracy
5. PacBio. (2022). Considerations for using the low and ultra-low DNA input workflows for whole genome sequencing. https://www.pacb.com/wp-content/uploads/Application-Note-Considerations-for-Using-the-Low-and-Ultra-Low-DNA-Input-Workflows-for-Whole-Genome-Sequencing.pdf
6. Pacific Biosciences of California, Inc. (2023, February 7). PacBio to Expand MAS-Seq Technology to 16S rRNA and Bulk RNA-Seq Solutions. https://www.pacb.com/press_releases/pacbio-to-expand-mas-seq-technology-to-16s-rrna-and-bulk-rna-seq-solutions/
7. Illumina, Inc. (2024). Deeper insights into the complex regions of the genome: Long-read sequencing helps resolve challenging regions of the genome. https://www.illumina.com/science/technology/next-generation-sequencing/long-read-sequencing.html