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

Companies are focused on making these platforms smaller and easier to use.

Fast forwarding to the near future and based on the recent past, sequencing instrument companies will continue to develop more user-friendly and cheaper technology, focused on the benchtop and clinical markets. Manufacturers will also continue to form partnerships and make acquisitions that place heavy bets on completely novel, potentially disruptive sequencing technologies.

On a worldwide basis, life science research along with drug discovery and development applications currently comprise the two largest sequencing markets. They together accounted for about $920 million in 2010 and are expected to reach nearly $1.7 billion in 2015 with a compound annual growth of 13%.

By far the largest market opportunity, though, is in emerging applications of personal genomics and clinical diagnostics. These segments are expected to account for $541 million by 2015 from $15.5 million in 2010, representing a CAGR of 103.5%.

Recent advancements in the field of next-generation sequencing have resulted in the advent of so-called personal genome machines (PGMs), smaller-scale, benchtop genome sequencers marketed by Illumina (MiSeq), Life Technologies (Ion Torrent), and Roche 454 (GS Junior). PGMs bring DNA sequencing directly into individual laboratories and will impact the high-throughput sequencing (HTS) market in the process. Companies will continue to advance these machines, but those aiming at the clinical space will need to gain regulatory approval for their use in clinical laboratory diagnostics.

Life Technologies’ Investments

A key disruptive technology introduced to the market in 2010 by Life Technologies is the Ion Torrent DNA sequencer. It has set a completely new competitive bar in PGMs. This technology eliminates the need for optical readout, instead gathering sequence data by directly sensing hydrogen ions produced by template-directed DNA synthesis.

In less than a year since Life Technologies first commercially launched its PGM, the semiconductor-based instrument became the best-selling sequencing machine in the world. The technology provides low cost and scalable sequencing on a massively parallel semiconductor-sensing device, or ion chip. Reactions are performed using all natural nucleotides, and the individual ion-sensitive chips are disposable and inexpensive. The instrument combines fluidics, micromachining, and semiconductor technology, allowing direct translation of genetic information to digital information.

The firm has ambitious plans for the magic sequencing machine. Last October, the company announced that it will seek FDA 510(k)-clearance for the Ion PGM™ sequencer in 2012 in order to expand it into the clinical setting. The company’s R&D plans will also certainly focus on kits to accompany this PGM, including its AmpliSeq™ Cancer Panel. It is the first product utilizing Ion AmpliSeq technology and covering oncogenes and tumor suppressor genes.

Additionally, Life Technologies has placed a big bet on another potentially disruptive technology, privately held Genia’s biological nanopore technology. In April 2011, Genia closed a strategic investment with Life Technologies. The technology comprises an engineered pore protein embedded in a lipid bilayer membrane. Single-stranded DNA (ssDNA) with its double-stranded end inside the vestibule of the nanopore and single-stranded end threaded through the transmembrane of the nanopore travels through the central pore of the protein.

As the ssDNA travels through the pore, it attenuates the current traveling through the membrane in a sequence-dependent manner, each of the four bases interacting with the nanopore recognition site differently and partially blocking the ion current by a specific amount characteristic of that base’s unique electrochemical interactions with the nanopore recognition site. DNA sequences are computed from the residual currents flowing through the nanopore/DNA complex.

Genia co-founder and CEO, Stefan Roever, commented that the platform can actively control the DNA template, moving it back-and-forth through the nanopore multiple times if needed. “We can oversample, rewind, and read again. You change the applied voltage and the DNA goes backward. If you capture the DNA in the pore, you can ‘dental floss’ it; you can read it 10–20 times.” Roever would not detail the read-out mechanism, other than to say, “our approach relies on some IT to reassemble those sequences.

“If Ion Torrent—electrical detection but requiring amplification—and Pacific Biosciences—single-molecule but optical—are third-generation sequencing technologies, then we’re fourth generation: single molecule, electrical detection,” said Roever. “That’s the holy grail because it combines low-cost instruments with simple sample prep. So we’d like to think of it as last-gen!”

Illumina’s Strategy

Illumina is planning to extend the clinical reach of its MiSeq for diagnosing infectious diseases. The firm says that through its November 2011 alliance with Siemens Healthcare Diagnostics, it will also apply the technology to identify potential treatment paths for these diseases. The companies said they plan to make existing Siemens molecular HIV tests compatible with MiSeq.

Illumina, like Life Technologies, has also put a stake in the nanopore sequencing space through its deal with Oxford Nanopore Technologies. Oxford is developing its GridION system, which uses nanopores for the direct, electronic analysis of single molecules including DNA, RNA, proteins, and other molecules. The company says that its nanopore-based method obviates the need for amplification or labeling by detecting a direct electrical signal.

While the company has not disclosed key elements of the system’s operation, a 2010 paper in Nature Nanotechnology described some important aspects of the process. The paper was published by Oxford scientists and their collaborators at the University of California, Santa Cruz (UCSC).

It describes the passage of ssDNA as it translocates through a protein nanopore. Movement of the ssDNA was controlled by polymerase-facilitated replication of individual DNA molecules and could be initiated under electronic control. Polymerase activity could be blocked in solution when the ssDNA was not at the nanopore opening, however, capture of the strand by the pore removes a blocking strand of nucleotides and allows the polymerase to function on the trapped strand.

Commenting on the technology, Jay Flatley, Illumina president and CEO said, “Oxford Nanopore’s technology holds tremendous promise to achieve the sub-$1,000 human genome. Making electrical measurements of unmodified DNA removes the need for complex sample prep and the high-performance optics found in today’s sequencing systems.”

Agilent’s Pipeline

Agilent will continue to enhance the efficiency of its next-gen sequencing technologies and has moved to augment its SureSelect technology platform. SureSelect is the company’s front-end method for isolating complex subsets used in targeted resequencing, for example. SureSelect, the company has explained, replaces other labor-intensive methods of targeted resequencing such as PCR techniques.

Eric Endicott, Agilent’s global public relations manager, life sciences group, told GEN that the company’s November 2011 acquisition of Halo Genomics’ technology will complement Aglient’s SureSelect target-enrichment platform technology. Halo Genomics’ HaloPlex technology, Endicott said, provides a high-performance solution for small capture sizes, at a speed that specifically addresses the needs of the desktop sequencing market.

In addition to expanding Agilent’s portfolio of solutions for the rapidly growing next-generation sequencing market, Halo Genomics’ technology further pulls Agilent’s target capture solutions toward the next-generation clinical sequencing market.

New Kid on the Block

Massachusetts 2010 startup Noblegen says its nanopore-based technology usually requires complex instruments, but has the potential to deliver high speed and low costs. Noblegen says its technology’s ability to directly and rapidly read DNA sequences could make it economically feasible to bring sequencing technology into clinical labs to diagnose cancer and other diseases.

Noblegen’s technology works by first converting genomic DNA into a synthetic version that’s labeled with four different fluorescent dyes, one for each type of base. Each base in the original sequence is represented by one fluorescently labeled segment in the synthetic one.

The synthetic sequences are then directly read out by Noblegen’s relatively simple instrument based on a silicon chip with pores a few nanometers in diameter and illuminated by an inexpensive laser. The long, charged synthetic molecules are pulled through the hole by electrostatic forces. As the DNA moves through the pore one segment at a time, the labels become detached, creating a flash of light that is then imaged by a CMOS sensor similar to those used in digital cameras.

NobleGen co-founder and CEO Frank Feist has said that the company’s goal is to aggressively drive down the cost and increase the speed of sequencing whole genomes to a point where it makes economic sense for hospital labs in the next three or four years.

While the company won’t divulge details of its current instrument prototypes, Feist noted that the technology could be scaled up to arrays of 400 by 400 nanopores that sequence over 500 gigabases an hour—or about one genome, covered 30 times, in 15 minutes.

All around, sequencing equipment manufacturers run some financial risk from spending cutbacks by research labs for this year. But all are focused on staying ahead of the technology curve through smaller, faster, and less expensive instrumentation. They are also counting on the future of clinical sequencing.

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

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