Patricia F. Fitzpatrick Dimond Ph.D. Technical Editor of Clinical OMICs President of BioInsight Communications

Oxford Nanopore and PacBio both have exciting technologies, but Illumina remains market leader.

Roche’s chairman, Franz Humer, said last week that the company has “other options” if its hostile takeover bid to acquire Illumina proves unsuccessful. “Illumina is not the only gene sequencing company, and there are other companies making quantum leaps in this field.

“Roche is also working on gene sequencing technologies of its own,” he further commented as Illumina continues to shun Roche’s $5.7 billion bid as “grossly inadequate.” Nonetheless, analysts believe that Illumina holds the lead in installed bases of sequencer instruments even as other companies introduce new technologies.

In a letter to Dr. Humer, Illumina’s Jay Flatley, president and CEO, articulated his company’s position. “Our history and track record of commercially effective innovation—combining game-changing technology developments with rapid product introductions—is unparalleled, and our current product portfolio, robust as it is, does not begin to fully reflect the value-creation potential of our research and development pipeline and the numerous ways in which we continue to push the envelope of genetic analysis scale and accessibility.”

In the high-stakes world of gene sequencing technology, “game changer” is becoming the adjectival phrase of choice, right up there with “disruptive,” as companies continue to introduce methods, instruments, and software platforms.

Nanopores Make Big Waves

The latest potential product entrant to win the appellation, according to company CEO Gordon Sanghera, is Oxford Nanopore Technologies’ MinION, set to go on sale in the second half of 2012. The company says the machine will cost less than $900. Way smaller than a bread basket, the MinION is about the size of a USB thumb drive.

The company described the device as well as its bigger sibling, the GridION system, on February 17 at the Advances in Genome Biology & Technology (AGBT) conference, which has emerged as the industry’s best-publicized hush-hush meeting at which to announce breakthroughs.

The MinION can be plugged into a laptop’s USB port. The GridION, a larger version of the machine, can be stacked to increase processing power. The sequencers are based on the company’s nanopore “strand sequencing” technology combined with the two electronic devices, GridION and MinION. The MinION, said to deliver 150 megabases of DNA sequences per hour, is intended for one-time use only and will sequence up to a million bases. According to some reports including a story in The New York Times, executives said that the GridION could cost about $30,000. However, an Oxford Nanopore spokesperson noted that this is incorrect as there is no fixed price for a single instrument.  The systems will reportedly be priced in packages that include instruments and consumables.

Oxford describes its technology as a method of DNA “strand sequencing” that uses an array of protein nanopores embedded in a polymer membrane. Each nanopore sequences multiple strands of DNA from solution in succession, as individual strands are passed through the nanopore by a proprietary “processive” enzyme. Base identification is accomplished by identifying characteristic electronic signals.

These signals are created by unique combinations of DNA bases as they pass through a specially engineered region inside the nanopore. DNA and enzyme are mixed in solution, engage with the nanopore for sequencing, and once the strand has been completed, a new strand is loaded into the nanopore for sequencing. The technology does not require sample amplification; the company says that any user-derived sample preparation that yields dsDNA will work with the system.

“If it does work, it will be a game-changer,” said Elaine Mardis, co-director of The Genome Institute at Washington University in St. Louis. Chad Nusbaum of the Broad Institute called it “impressive, credible, possibly amazing,” according to an article in The New York Times.

But does it work? Life Technologies’ Ion Torrent division head, Jonathan Rothberg, and inventor of the ion torrent technology behind that company’s Personal Genome Machine, said in an e-mail interview with Forbes’ Matthew Harper, “I don’t believe it.” If it is real, Rothberg said, it has to scale a million-fold, from 5,000 bases to six billion. “How are they going to do that all this year? How are they going to manufacture the parts?”

Rothberg noted that Life Technologies showed three human genomes completed with the Ion Proton instrument last month at AGBT. More importantly, he added, they had the machine—not a mock-up or a design—on the stage. “That’s where you need to be to ship mid-year.”

Rotherberg’s technology combines fluidics, micromachining, and semiconductor technology, allowing direct translation of DNA into DNA sequence. The technology works by generating sequencing data by directly sensing hydrogen ions produced by template-directed DNA synthesis.

In less than a year since the semi-conductor-based instrument was launched, it became the best-selling next-generation sequencing machine in the world. The technology provides low cost and scalable sequencing on a massively parallel ion chip. Reactions are performed using natural nucleotides, and the individual ion-sensitive chips are disposable and inexpensive.

Eavesdropping on a Single DNA Polymerase

Other breakthrough technologies for DNA sequencing are in the works. For example, PacBio SMRT (single-molecule, real-time) technology incorporates single-molecule sequencing techniques and advanced analytics to reveal true biology in real time. The company reports that the technology can perform long reads, and produce fast results and more informative data at lower overall costs.

PacBio’s SMRT technology platform depends on innovations that overcome major challenges facing the field of DNA sequencing. These include its SMRT Cell, which enables single-molecule, real-time observation of individual fluorophores against a dense background of labeled nucleotides while maintaining a high signal-to-noise ratio. Another key innovation is the use of phospholinked nucleotides, which enable long read lengths by producing a completely natural DNA strand through fast, accurate, and processive DNA synthesis.

Company founder and chief technology officer Stephen Turner, Ph.D., told GEN that the company’s technology is differentiated by several features from other current sequencing technologies. “It’s the first real-time technique to reach the marketplace,” Dr. Turner said. “This means we can get longer read lengths, longer than any step and repeat systems, allowing read lengths of on average from 2,500 to 3,000 bases and up to 10,000 bases in little over an hour. For much of the installed base of systems, it would take several days to look at several hundred base sequences, requiring a multiday protocol.”

Dr. Turner also said that SMRT sequencing, because of longer read lengths, more often detects miss-mapping because of the larger amount of gene sequence context the method enables. “Another advantage is the richness of data the technology provides. Ours is the first system that provides information about chemical modifications of DNA bases such as methylation and, for example, 5-formyl and 5-carboxy cytosine, two recently discovered modifications.

“You can detect these modifications with mass spec or HPLC, but these are not high-throughput techniques that give you the genomic context along with the sequencing information,” Dr. Turner pointed out. “The PacBio sequencer puts those modifications into the context of a sequencing assay, making it more adaptable to whole-genome studies.

“We are finding an ever-increasing alphabet of bases that serve important biological functions,” Dr. Turner noted. “Expanding the accessible genome alphabet from four letters—A,G,T,C—to the plethora of chemically modified bases that occur in the genome provides a new vista onto the true nature of important systems in epidemiology, development, and cancer.”

PacBio has had its challenges, though, since it started shipping systems in April 2011. In September 2011, the company said it planned to reduce its headcount by 130 employees, 28% of its staff. Its operations and R&D segments were most affected. The announcement resulted in PacBio’s stock droping by 31% in one day and by more than 75% since the company’s  IPO in October 2010. The company said the layofffs were the result of uncertainties associated with the econonomic environment and were made to better position the company for long-term success.

PacBio wasn’t the only company to suffer from predictions of lower federal funding for large instrumentation purchases. Complete Genomics experienced a 35% decrease in stock price, and Illumina shares dropped 18% in July 2011 after the firm offered a revenue and earnings-per-share forecast that fell below Wall Street expectations.

Meanwhile, Illumina remains the dominant marketer of DNA sequencing instruments and owns a 15% stake in Oxford, although it doesn’t have marketing rights to Oxford’s nanopore device. While it is clear that there are other players in the field, Illumina will likely remain the object of Roche’s desire. Its market share is a huge advantage, and Roche would be better able to leverage its own expertise in diagnostics and facilitate its foray into personalized medicine with Illumina under its wing.

Patricia F. Dimond, Ph.D. ([email protected]), is a principal at BioInsight Consulting.

This article was updated on March 14 to include Oxford Nanopore’s clarification about the pricing of its GridION instrument.

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