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ZS Genetics Gets Ready to Sequence DNA; Bets on Long Reads, Low Consumable Cost

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ZS Genetics is hoping to leverage recent technical developments it has made to sequence DNA later this year, using its transmission electron microscopy-based single-molecule technology.
 
The North Reading, Mass.-based startup hopes that read lengths of several kilobases, coupled with a throughput similar to current second-generation sequencing platforms, will persuade potential customers to go with its technology, which will require buying a pricey piece of equipment but promises very low running costs.
 
“One of the overwhelming selling points of the ZS approach is going to be sequence length,” said Kelley Thomas, co-director of the Hubbard Center for Genome Studies at the University of New Hampshire and a ZS Genetics collaborator and scientific advisory board member. “It’s an inherent property of the approach that allows the sequences, potentially, to be captured in very long stretches.”
 
Since it was founded in 2005, ZS Genetics has been developing a method that uses a transmission electron microscope to directly image double-stranded DNA molecules (see In Sequence 2/20/2007). Because DNA is normally invisible to TEM because of poor contrast, the company generates DNA where most of the bases contain heavy atoms. These bases appear in the electron micrograph as black dots of different sizes, depending on the label.
 
The labeled nucleotides are incorporated in an in vitro synthesis reaction using undisclosed polymerases. Although the company has enough different labels, which include off-she-shelf ones like bromouridine, to cover all four bases, the plan is to use three different labels initially and leave one base unlabeled, according to president and founder William Glover. He and his colleagues have already prepared DNA with three labels but have not yet sequenced it.
 
The length of the modified template DNA will determine the read length of the system. Based on their initial results, Glover said he is confident that the company will be able to reach a read length of 5 to 8 kilobases, though he would like to extend this to 10 to 12 kilobases.
 
Thomas’ group has been helping the company generate the labeled DNA, and he thinks the labeling step will not be limiting. “It turns out that the incorporation of modified nucleotides is not so difficult,” he said. “Most of the commercially available polymerases are not bad at doing this.”
 
What has proven more tricky is to manufacture a thin enough substrate on which the labeled DNA is placed prior to TEM imaging. Developing this substrate, which is mounted on a silicon chip and is essentially transparent to the electron beam, “was a lot more work than we thought it was going to be” and required company researchers to make “a lot of small changes” to standard semiconductor manufacturing techniques, Glover told In Sequence last week.
 

“One of the overwhelming selling points of the ZS approach is going to be sequence length.”

Although they have already managed to reduce the substrate’s thickness from 100 nanometers to 11 nanometers, they need to go down to 7 nanometers in order to obtain good DNA images.
 
While the company is nearly finished developing the substrate, it is only “halfway through” increasing the density with which it can mount DNA strands on the substrate that are suitable for imaging. The undisclosed process involves a fluidic flow to separate and stretch individual DNA molecules on the slide.
 
“We have no problem doing them at low density where you see one molecule at a time,” Glover said, “but getting them to a higher density, which gives you much higher throughput, without tangling them up is something we are still working on.”
 
The density of DNA molecules on the substrate will determine the initial throughput of the system, which Glover expects to be on the order of tens of megabases per hour initially, or “very comparable” to the current second-generation sequencers. Also, the run time is not fixed and will depend on the number of images taken and analyzed.
 
Ready to Sequence
 
With labeled DNA templates and suitable substrates soon in hand, ZS Genetics plans to start sequencing this summer, in collaboration with early-access users. These partners will label and mount DNA samples according to the company’s protocols, and use microfluidics equipment provided by ZS Genetics. The company will analyze the samples.
 
Glover said the firm is currently “in active discussions” with undisclosed early-access customers and is looking for additional “top labs in the world” to get involved. “We want feedback on the entire thing; we want the customers to do the lab steps themselves and tell us if this is what they want to be doing routinely, or is there something wrong with it?”
 
These experiments will also allow the company to determine the sequencing accuracy, a big unknown at the moment, and to develop its automated image-analysis software.
 
Following the testing phase, which Glover expects to take at least six months, ZS Genetics plans to commercialize the system.
 
It will consist of a modified TEM, the price of which could range from $1 million to $3 million, as well as equipment to prepare the DNA samples. ZS Genetics will also provide reagents, slides, and software to provide base calls and quality scores.
 
“The key thing is, the stuff we are providing, except for the microscope, is not complicated stuff,” Glover said. And while the instrument will cost a lot, consumables will be very cheap.
 
In marketing material, the company estimates that generating a high-quality, fully-assembled human genome will cost less than $70,000 on the instrument in “fully weighted running costs” that include amortized capital costs.
 
Since consumables will be cheap, ZS Genetics plans to “make a little money on everything,” including initial systems sales, consumables, and digital camera upgrades. “We are not trying to make outrageous amounts of money,” Glover said. “The whole idea is that the total amount spent on sequencing has to go down.”
 
The main competitors for ZS Genetics’ EM technology, if it yields usable sequence data, will likely come from other companies developing single-molecule long-read technologies, such as Pacific Biosciences and VisiGen Biotechnologies.
 
“Whoever can make it work would have a dramatic effect on our ability to sequence individual humans,” Thomas said.
 
“The issues around read length ... really come into play when you start talking about complex eukaryotic genomes, where size, and the number of repeats, and the amount of gene duplication that’s going on in the genome really start to become a problem.” Though paired-end reads could help address this problem, “to be able to sequence several thousand nucleotides at a time in a stretch will help,” he said.
 
His own research involves mutation analysis in model organisms, and he has used both 454’s and Illumina’s sequencing technologies as part of collaborations. The reason for starting the collaboration with ZS Genetics was that his work “directly benefits from sequencing and would directly benefit very much from the development of this,” Thomas said.
 
In addition, part of mission of the Hubbard Center for Genome Studies is to “develop intellectual capital in the region,” he said, and the collaboration, funded with a small grant from ZS Genetics, gives undergraduate students a “great opportunity” to get a peek into industry.
 
In addition to using lab space at the University of New Hampshire, ZS Genetics, which has eight full-time employees, now has its own lab in Danvers, Mass., where its electron microscope is housed.
 
Since February of 2007, ZS Genetics has increased its total amount of funding from private investors to $3 million from $1.8 million and is currently raising another $1.5 million. “That will probably be our last major round” before finishing developing the technology, Glover said.

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