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Taiwanese Team Explores New Sequencing Chemistry to Power Novel Single-Molecule Platform


NEW YORK – A DNA polymerase from Thermococcus sp. has properties that could power the chemistry of a new single-molecule sequencing platform, according to new research by a team in Taiwan.

The study, published in June in Nature Communications Biology, is the result of a three-year collaboration between Ming-Daw Tsai's group at the Taiwan Academia Sinica's Institute of Biological Chemistry and Personal Genomics, a company that is trying to build a next-generation sequencing platform using semiconductor chips for optoelectronic signal detection.

Specifically, the 9-degree-N DNA polymerase possesses both the ability to incorporate nucleotide analogs containing terminator groups, which are used in sequencing-by-synthesis methods such as Illumina's, as well as 3'-esterase activity to remove the 3'-terminator group. By incorporating fluorophores at the 3' position, the enzyme could thus remove the blocker and release the base-associated signal all at once, speeding up the reaction.

"The esterase activity is the key," said Tsai. "It's a special property of this DNA polymerase that makes it cleave the esters and that leads to a pause that allows detection."

Personal Genomics CEO Johnsee Lee, who is also an author of the study, said the sequencing chemistry was just one of several his firm is exploring for use with its hardware. "There are many different versions of 3' chemistry and many different ways to play with it, depending on which is most compatible with the detection system," he said.

Michael Metzker, an expert on sequencing-by-synthesis and a professor at the Baylor College of Medicine Human Genome Sequencing Center, said the chemistry described in the paper could be useful for a single-molecule sequencing approach but that he wanted to see more studies. "There needs to be a lot more work done to convincingly show this would work in a sequencing method," he said. Other researchers have already tried and failed to implement many of the strategies the authors claim to have succeeded at, he said.

Tsai noted that the paper was meant as proof-of-concept and agreed that optimization, of the enzyme and other aspects of the chemistry described, would be required before it would be ready for a commercial product.

Taiwan-based Personal Genomics is trying to build a new single-molecule sequencing platform that is faster, cheaper, and more compact than Illumina's current short-read technology, according to Lee. The firm also wants to provide longer reads than Illumina, of more than 1,000 base pairs.

"Sequencing is too expensive at the moment," Lee said. "We want to make sure it's a cost-effective platform." Lee said the firm is looking to use Taiwan's expertise in semiconductor chip manufacturing to improve detection methods. It also collaborated with Tsai and others to explore a new sequencing chemistry that would be compatible with its hardware.

"We have both technologies and we're putting them together," Lee said. "Everything is on a semiconductor chip."

The firm has in-house teams working on the chemistry, optics, and semiconductor chips, but also has outsourced many aspects of its technology development, Lee said.

The challenge is to develop a detector sensitive enough for a weak single-molecule signal, which is approximately 20 to 30 photons per millisecond, Lee said. "The difficulty is huge compared to a regular sensor," he said. And that's only half the equation.

In their paper, the researchers noted that some publications reported 3'-esterase activity in T7 and Taq polymerases as early as 1995, but that follow-up studies had not been conducted.

"Back in the 90s, when folks were really starting to think about this for the first time, there were a number of patents that describe having the blocking group have the fluorescent dye attached," Metzker explained. "It's a sexy approach." With 3'-esterase activity, one could "remove the blocking group and dye in one swoop."

In theory, that's "way more effective" than the cyclic reversible terminator approach implemented by Illumina and others, he said, where the fluorophore is connected to the 5' end and the reaction must pause to cleave the blocking group, cleave the fluorophore, and restore the 3'-OH group to enable the next round of nucleotide incorporation.

"But in our method, the dye is attached to the ester group, the 3'-hydroxyl group," Tsai said. "When the enzyme cleaves the ester, it also cleaves the dye," reducing the number of reaction steps.

The researchers reported their chosen enzyme could incorporate terminator nucleotide analogs, cleave the 3'-OH group, and that such activity was intrinsic to the enzyme. They also said they could get reads of more than 400 base pairs.

Metzker, however, said he was not fully convinced by the published study. First, the enzyme described is also commercially available from New England Biolabs, dubbed Therminator. "I'm not sure why they're not calling it what it is," he said.

More importantly, he said, "the data aren't completely compelling because the purity of the modified nucleotides can sometimes blur what the interpretation is." The authors reported the purity of their modified nucleotides was greater than 99 percent. But Metzker said contamination by natural nucleotides could make it look like the polymerase was removing the blocking group, when it was never actually there. "You need [the natural nucleotides] to be almost non-existent," he said. "They always compete [for incorporation] and usually will win."

Additionally, other researchers have tried, and failed, to achieve incorporation of nucleotides with the dye attached to the 3' position. "The active site, where nucleotides bind, is really crowded and it's too difficult to incorporate them when you have a bulky group," Metzker said.

"They don't even show they're getting base-specific incorporation," he added, and instead "just kind of conclude they're getting long reads."

In response to Metzker's criticisms, Tsai and Lee suggested that they're aware of these issues and that their data back up their claims.

"We have used both mass spectrometry and X-ray crystallography to show that contamination was not an issue in the first couple of cycles," Tsai said.

He also acknowledged that the team used the same dye for all the different nucleotide analogs, instead of a different dye for each one. "This will be an important next step, but only one of many important steps that remain to be optimized before commercialization," he said. "The reason we did not use different fluorophores for each [nucleotide analog] is due to sample availability, not a technical issue. A more important issue in terms of proof of concept is the error rate, which is reasonably good in our system."

Lastly, Metzker noted that the incorporation rate was slow, which would make the entire platform slow compared to other single-molecule platforms, for example from Pacific Biosciences and Oxford Nanopore Technologies. The read length, even at 1,000 base pairs, would also pale in comparison to reads from those other platforms.

Lee suggested that Personal Genomics, if successful, would be cheaper than PacBio. But most often, he compared the technology he's working on to Illumina's.

Meantime, Personal Genomics continues to search for the right combination of chemistry and hardware. Lee said the firm is gearing up for a product demonstration "maybe sometime in the first half of next year."