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LaserGen Says Its New Reversible Terminators Could Improve Several Sequencing Platforms

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Two years after publishing its first reversible terminator nucleotide, sequencing technology company LaserGen has developed a full set of the reagents that it claims could improve a variety of existing and impending second-generation sequencing platforms.

The Houston-based startup (see In Sequence's sister publication, GenomeWeb Daily News, 10/16/2006) plans to commercialize the reagents, called Lightning Terminators, for use with at least one existing sequencing platform. Long term, it wants to develop its own sequencing instrument for use in personalized genomics.

Last month, at the Advances in Genome Biology and Technology conference in Marco Island, Fla., LaserGen founder and CEO Michael Metzker presented results from the company's development efforts over the last couple of years.

The company's dye-labeled, photo-cleavable reversible terminators differ from many others in that they have an unblocked 3'-OH group, which enables researchers to use them in sequencing reactions with off-the-shelf polymerases.

A single light-induced cleavage step removes the terminating group as well as the dye and leaves a naturally occurring base. The company has also been working on chemically cleavable versions of the terminators.

Kinetic experiments in collaboration with researchers at New England Biolabs have shown that the Lightning Terminators have an "efficiency value" — a combination of how well they bind to the polymerase and how fast they get incorporated — that is better than that of natural nucleotides, according to Metzker, who is an associate professor at Baylor College of Medicine's Human Genome Sequencing Center.

Even though the researchers have to unblock the growing DNA chain after each cycle, using UV light, a polymerase incorporates the terminator nucleotides at a rate of 15 bases per second, faster than Pacific BioSciences' real-time sequencing technology at the moment, Metzker pointed out.

In addition, the polymerase makes fewer mismatch errors using Lightning Terminators than using natural nucleotides, he said.

He and his colleagues have been using Vent, a wildtype polymerase, and Therminator, a wildtype enzyme with a single mutation, to test the reagents, and "both tend to show the same kind of performance with these Lightning Terminators."

Illumina's Genome Analyzer and Intelligent Bio-Systems' pending Pinpoint Sequencer also use reversible terminators, but both systems' nucleotides have a blocked 3'-OH group and a dye that is attached to the base through a linker, which leaves a "molecular scar" when it is removed, Metzker said.

Helicos BioSciences' "virtual terminators," on the other hand, have an unblocked 3'-OH group like LaserGen's reagents, and also require a single cleavage step to remove both the dye and the inhibitory group. But since the terminating group is attached through a linker, they retain a "molecular scar," unlike LaserGen's terminators, according to Metzker.

The Lightning Terminators build on work that Metzker and his colleagues published in 2007 in Nucleic Acids Research. In that paper, they showed their first 3'-unblocked reversible terminator, which has a terminating 2-nitrobenzyl group attached directly to the adenine base.

When they tried to use the same strategy to build another terminator, attaching the 2-nitrobenzyl group directly to thymine, they found that the resulting nucleotide was universal and no longer paired with adenine.

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As an alternative, the researchers added the terminating group to the hydroxyl-"handle" of naturally occurring modified versions of the pyrimidine bases, hydroxymethyluracil and hydroxylmethylcytosine. The resulting reversible terminators "perform extremely well," according to Metzker.

Since then, he and his colleagues have been refining the structure of the terminating 2-nitrobenzyl-group in order to obtain "exquisite termination" for all four reversible terminators, he said, "and in that process, we discovered it also gave us better discrimination against mismatch bases, while not affecting the binding constants or the incorporation rate."

At the moment, the scientists are setting up sequencing experiments to demonstrate what read length, accuracy, and other parameters they can achieve with their Lightning Terminators, so "we can really reduce it to sequencing metrics that everyone understands better," Metzker said.

LaserGen, which amplifies DNA for sequencing on a home-grown sequencer by emulsion PCR on beads, said that within the next few months it plans to sequence E. coli and other bacteria that the large genome centers have used in the past to test second-gen platforms made by Illumina, 454, and Applied Biosystems.

"We had a little bit of a learning curve with the imaging [and] the basecalling, but we are moving to [the bacterial-sequencing] experiment as we speak," he said.

In order to help commercialize the Lightning Terminators and raise money, LaserGen, which currently has eight full-time employees, hired a vice president of business development last fall.

Up until now, the company has been mainly funded through NIH grants as well as a research collaboration with an undisclosed large sequencing company. The company's goal is to raise $2 million this year from angel investors or venture capital firms.

According to Metzker, owing to their properties, the new reversible terminators could benefit a variety of existing sequencing platforms, and LaserGen is currently discussing potential commercialization options with several companies, including both existing sequencing vendors and several large undisclosed companies that "want to get into this space but don't have existing platforms."

LaserGen has designed its reagents to work with four-color sequencing systems like Illumina's GA, ABI's SOLiD, or George Church's Polonator, and has created unlabeled versions to work with other systems, such as 454's pyrosequencing chemistry.

In addition, Metzker said, the Lightning Terminators could be used in single-molecule sequencing on platforms such as Helicos' or PacBio's.

LaserGen's first choice would be to partner with existing sequencing vendors that have a platform in the market, said Metzker. "We would at least like to see two different platforms using this chemistry," he said. "If we had our choice, that's how we could see this chemistry having the greatest benefit to the community."

Long term, LaserGen plans to develop a sequencing instrument of its own, using the four-laser Pulsed Multiline Excitation imaging technology that Metzker developed several years ago. That instrument would focus on applications in personalized genomics, he said.

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