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New Pacific Biosciences Revio Chemistry Promises Lower Sample Input, Sequencing Costs

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NEW YORK – Pacific Biosciences on Tuesday launched a new sequencing chemistry for its Revio platform that promises to reduce DNA input requirements and costs while boosting data output for HiFi long-read sequencing.

Dubbed Sprq, the updated chemistry, along with improved data processing, will reduce sequencing costs for a 20X human genome to under $500, half the current cost using the Revio system, according to the firm. "The HiFi data you see will be very consistent with the existing HiFi data, but you get more of it while using less starting material," said PacBio CEO Christian Henry.

DNA input requirements are reduced fourfold with Sprq through more efficient sample loading onto to the SMRT flow cell.

According to Aaron Wenger, a PacBio product manager, DNA reaches a zero-mode waveguide (ZMW), a nanoscale well within the flow cell where the sequencing action happens, via a two-step process: The molecule first diffuses within a column of liquid to the SMRT cell surface, followed by lateral movement along the surface into a ZMW.

With the current chemistry, Henry said, the "vast majority" of DNA is lost in the loading process, and less than 1 percent of DNA molecules make it to a ZMW. Sprq improves that rate by making sure more DNA molecules reach the SMRT cell surface, Wenger noted, but did not provide details on the mechanism.

As a result, Sprq requires just 500 ng of DNA compared with 2 μg using the existing chemistry. "That is a major breakthrough for us," Henry said. "It fundamentally changes the number of samples that are accessible with PacBio sequencing."

Besides lowering input requirements, Sprq also boosts the raw base accuracy for HiFi sequencing "pretty significantly" by incorporating an optimized version of the Phi29 DNA polymerase, he noted.

According to Wenger, the company's enzymology team produced and screened tens of thousands of polymerase variants, and the new enzyme used in Sprq improves accuracy and polymerase survival.

Taking all the improvements together, the new chemistry promises to increase sequencing yield by 33 percent, which "dramatically changes the economics," Henry said. Using Sprq, researchers can obtain two human genomes at 20X coverage per SMRT cell, adding up to eight genomes per Revio run, as the instrument accommodates four flow cells. This doubles the number of genomes achieved by the old chemistry on the Revio.

The Sprq chemistry still uses the same protocols and flow cells as its predecessor but will have "slightly different reagents," Henry said. Sequencing run time also remains the same, at 24 hours.

With the same list price as the existing chemistry, Sprq effectively cuts sequencing costs for a 20X HiFi genome in half, from $995 to below $500, Henry noted, while doubling the output per run. Customers with high volumes will get "reasonable discounts," he added.

PacBio also released a SMRT Link software upgrade that promises to augment multiomic analysis capabilities for Revio runs. The updated software comes with improved accuracy for 5mC calling, Henry said, and adds a new capability to call 6mA for the Fiber-seq chromatin accessibility assay.

Alexander Hoischen, a researcher at Radboud University Medical Center in the Netherlands, called the updates "very attractive." His group currently operates four Revios, three of which are dedicated to a prospective clinical utility study of HiFi sequencing as a first-line test for rare diseases.

Since his team primarily works with blood samples from rare disease patients, input requirements are rarely an issue, Hoischen noted. Still, the lowered DNA input will make sample prep automation easier, he said.

While Henry tried to make the case that a 20X HiFi genome is "more than equivalent" to a 30X short-read sequencing genome, it remains to be seen whether researchers will accept this notion.

Hoischen said his team currently sequences one genome per SMRT cell at 30X coverage. However, a 20X HiFi genome "is already extremely good," he said, based on data from a recent study that he and his collaborators, including researchers from PacBio, conducted, and he is considering 20X genomes down the road.

Although Hoischen is "extremely excited" about the reduced cost of sequencing, he noted that "we need to take that with a bit of a grain of salt" as costs can fluctuate in the real world. Also, the list price does not reflect the cost of library preparation and quality control, which has totaled about $100 per sample for his team's studies.

PacBio's improvements with the new chemistry "are all very positive things," said Anoja Perera, director of sequencing and discovery genomics at the Stowers Institute for Medical Research. "We have really been wanting a way to start with small amounts of material, so it is really great to hear they will be releasing a product where we can start with a lower input amount."

Perera's core lab installed a Revio platform in July and has used it so far for RNA and genome sequencing and methylation detection, as well as single-cell isoform analysis. In addition to the lower sample input, Perera said she is "super excited" about the reduced cost, noting that her lab currently performs many RNA sequencing experiments using short reads mainly because of cost. "With short-read sequencing, you are only getting part of the picture," she said.

Echoing Hoischen's point, Perera noted that costs quoted by companies are typically a best-case scenario that sometimes does not translate into the real world. For instance, her lab has struggled to utilize sequencing consumables before their expiration date, leading to reagent waste and higher experiment costs. For example, a Revio plate contains reagents for sequencing four SMRT flow cells and is only good for around 300 hours after first usage, and her lab sometimes does not have enough samples to run all four flow cells within that time frame. 

PacBio said the new Sprq chemistry and SMRT Link software are available for order immediately and will begin shipping in December. Henry said the company currently does not have a plan to phase out the existing chemistry and will continue to support it "for as long as customers need it."

"We do have further advancements in research and development right now, both on the platform front and on the chemistry front, that will take costs even below where we are today," Henry said. "I would not be surprised to see a day where long-read sequencing is not very different in costs from short-read sequencing."