Developers of next-generation and third-generation sequencing technologies compete for first place in the genomics marathon.
The field of DNA sequencing has come a long way since the first Sanger sequencing method became available in the 1970s. The first publication of this method reported the sequencing of 24 base pairs of DNA—a major milestone.
Sequencing technologies steadily became more efficient in the 1980s. When the International Human Genome Project was initiated in 1990, the race to improve sequencing technology truly began in earnest. The project took 13 years to complete and cost nearly $3 billion to sequence a single human genome, which is too slow and too costly by today’s standards. Since the project was completed in 2003, next-generation sequencing (NGS) technologies have been popping up left and right, with each emerging technology claiming to sequence a human genome (or multiple genomes, simultaneously) cheaper, faster, and with greater accuracy.
Despite the emergence of non-Sanger methods, the rich legacy of the Sanger sequencing method lives on with Beckman Coulter’s (Fullerton, Calif.) GenomeLab platform. Edna Betgovargez, senior strategic marketing specialist in the Chemistry, Discovery, and Automation Business Group at Beckman Coulter, helps support the GenomeLab GeXP genetic analysis system. According to the Beckam Coulter Web site, the Genomelab GeXp is an all-in-one genetic analysis system capable of performing quantitative gene expression, genome sequencing, fragment analysis, single base extension genotyping, and more.In addition, this Sanger sequencing-based method has a read length of 700 bases per sample and 98% base-calling accuracy in about a 100-minute cycle. “What is really unique about our system is that we use only one gel, one array, one software package, and one instrument to do everything,” says Betgovargez.
As with all Sanger sequencing methods, the GenomeLab GeXP genetic analysis system utilizes a dye terminator technology (Beckman Coulter’s WellRead dyes). And here’s how it works. Following incubation of the PCR-amplified genomic fragment sample with the terminator dyes, each terminated reaction sample is separated via capillary electrophoresis. “With [capillary electrophoresis], you move the analyte in an electric field. You have a sieving gel that then discriminates between the different parts in order to do the sequencing,” says Hans Dewald, also with the Chemistry, Discovery, and Automation Business Group at Beckman Coulter. The capillary electrophoresis system is then coupled with a fluorescence laser detector that “reads” each dye, generating a peak that an analyzer interprets as a different DNA nucleotide base. Through bioinformatics software, one can overlay all of these peaks and assemble information from each fragment to create a genomic map of the sequenced region.
At Agencourt Bioscience (Beverly, Mass.), a Beckman Coulter company, a number of DNA sequencing services are provided to pharmaceutical companies. Says Douglas Smith, PhD, director of science and technology at Agencourt: “The reason that most pharmaceutical companies like to outsource is because it is expensive to run the platforms.” He adds that, in addition to purchasing the platform, pharmaceutical companies would need to set up a laboratory; train employees on all of the current, as well as rapidly emerging, platforms and applications; and deal with the massive data storage and processing challenges that are inherent in genome sequencing projects notorious for generating terabytes of data.
Ready for the clinic?
Although Beckman and Agencourt are major players in the NGS space, the story of NGS technology really starts with Roche. Their platform, called Genome Sequencer FLX was the first NGS technology, and was developed in partnership with one of their recent acquisition companies, 454 Life Sciences (Branford, Conn.). This pyrosequencing-based platform currently has a 500-base pair average read length and is a well-established research tool. However, according to Tim Harkins, PhD, marketing manager for genome sequencing at Roche in Indianapolis, Ind., the company is now pushing its Genome Sequencer FLX platform into the clinic, where it has the potential to become an even more powerful translational medicine tool.
This trend was evident in presentations by researchers who used the Genome Sequencer FLX platform at the 2009 Annual Meeting of Advances in Genome Biology and Technology (AGBT). Sequencing of highly polymorphic and biologically-relevant regions of the human genome was a major theme. Among the topics presented were the sequencing of human leukocyte antigen genes, which are critical in organ transplants, and the sequencing of VDJ genes, which code for antibodies. Also at the AGBT conference, Roche reported that the Genome Sequencer FLX platform was used to sequence HIV genomes in an effort to identify drug-resistant mutations, which could lead to better therapeutic options.
Consistent with the theme of bringing emerging DNA sequencing technologies into the clinic, scientists at Navigenics (San Francisco, Calif.) want to bring the world of personalized medicine into the future by bringing into the physician’s office table-top devices that translate a patient’s genome sequence into actionable information that allows a physician to make medical decisions within a matter of minutes. “We take a DNA sample from an individual early in his life, extract all of the heritable risk information from that genome sequence, put all of that info into a big computer, and then we get a rank ordered list of your diseases,” says Dietrich Stephan, PhD, founder of Navigenics. “It’s just software that sits atop of your genome sequencer and extracts the relevant info and assigns risks to it; the software has the interface for the physician that makes all of this very complicated info simple, so they can act on it.” This software is commercially available.
In an article published in the November 6, 2008 issue of Nature, Illumina Cambridge (UK) reported that using their NGS platform they were able to sequence one human genome for $100,000 in two months, employing the efforts of 150 people, which seems quite a rapid advancement for NGS technology. However, says Peer Staehler, chief scientific officer and vice president of global marketing at febit (Lexington, Mass.): “While NGS has advanced a lot, it still is far from being something that can be used in a reasonable manner at high throughput.” Staehler explains that such low throughput is due to the fact that current NGS technologies “cannot target a particular part of the genome, [they] can only sequence the whole thing.” And that’s exactly what febit’s new HybSelect technology does—it allows users to select for (capture) regions of interest from whole genomes.
HybSelect’s workflow is as follows. Genomic DNA is first processed by fragmenting it ultrasonically (or by nebulization) and then adapters are attached for downstream processing. The fragments, which currently range from one to 10 megabases, are then hybridized with febit’s customized microfluidic DNA biochip, which captures the region of interest and allows it to be enriched and purified prior to downstream sequencing.
At the AGBT meeting, Agilent Technologies (Santa Clara, Calif.) launched a new product that promises to enable researchers to accomplish the same goal. The product, called SureSelect Target Enrichment System, is customizable, allowing researchers to pre-design the genomic regions they plan to target. “Potentially what the technology does is it allows researchers to just look at the areas of interest in the genome and discard the rest, which allows them to do a lot more sequencing that is useful for less money and a lot faster,” says Fred Ernani, PhD, senior product manager of emerging genomic applications at Agilent. Ernani estimates that with a 300-fold enrichment in target sequence, there will be a 300-fold reduction in the cost of sequencing that target.
The method for SureSelect technology, which is being optimized to work in front of many commercially-available NGS platforms, was licensed from the Broad Institute (Cambridge, Mass.), explains Emily LeProust, PhD, manager of chemistry development at Agilent: The technology involves hybridization, in solution, of the customer’s prepared genomic DNA sample with the kit’s customer-designed, biotinylated RNA capture probes. Streptavidin-coated, magnetic beads are then used to remove the hybridized target sequence from non-targeted sequence; the target sequence is subsequently amplified and sequenced.
The next generation
In addition to developers of NGS and NGS-associated technologies, a new group of sequencing technologies recently entered the genome sequencing race; they are called third-generation sequencers. Pacific Biosciences (Menlo Park, Calif.) is currently developing a third-generation DNA sequencing technology that is positioned for commercial release in 2010. But, what makes third-generation DNA sequencing platforms different from their second-generation/NGS predecessors? According to Stephen Turner, PhD, chief scientific officer of Pacific Biosciences, the main difference is that the younger sequencing technology is trying to increase the read length and accelerate the read time that NGS technologies sacrificed in an attempt to improve throughput over traditional Sanger sequencers. The read length feature on any sequencer is defined as the number of consecutive bases out of a genome that a technology can provide before it faces limitations, such as a low resolution, that can cause inaccurate base calling. The longer the read length, the more accurate the genomic map.
At the recent AGBT conference, Pacific Biosciences reported that their third-generation sequencing platform has a read length of approximately 1,000 bases, with some instances of within-run maximums of 3,000-base read lengths.
Another human-only, third-generation sequencing platform is produced by Complete Genomics (Mountain View, Calif.). Complete Genomics’ technology “employs high-density DNA [glass slide] nanoarrays populated with DNA nano-balls (DNBs), and uses a non-sequential unchained read technology called combinatorial probe-anchor ligation or cPAL; both technologies reduce reagent consumption and imaging time,” says company president and chief executive officer, Clifford Reid, PhD. Like other third-generation sequencing technologies, these innovations allow genome sequencing to be performed at a higher throughput and at a lower cost than second-generation (NGS) methods.
Complete Genomics expects to use their platform to provide a commercial sequencing service to the pharmaceutical and biotechnology industries, following their commercial launch in June 2009.
Also based on the ligation of the clonally-amplified genomic DNA fragments is Applied Biosystems’ SOLiD sequencing platform. However, in contrast to Complete Genomics’ platform, the amplified DNA is linked to beads, not a glass glide. A major distinction in the SOLiD platform’s principle is its “two base encoding” technology, which allows the user to attain a greater than 99.94% base calling accuracy, according to Kevin McKernan, distinguished scientific fellow at Applied Biosystems, a division of Life Technologies Corp., Beverly, Mass.
“The new SOLiD 3 System offers several enhancements, including higher bead density, walk-away automation, data analysis tools, and multiplex capability,” says McKernan. He explains each enhancement, separately. “In Applied Biosystems’ R&D Labs, higher bead density has yielded throughput of up to one billion tags and 40 gigabases per run. Walk-away sample loading automation, combined with application-specific data analysis tools streamline sample processing and reduce hands-on time. Multiplexing provides users with greater scalability to conduct small pilot or large genome-wide studies on the same system.”
In summary, the race to develop a sequencing technology that can sequence the human genome the fastest and cheapest is still on. With each emerging technology, the race gets tighter, generating a healthy competition that will likely last for some time, as each of these technologies has unique features that transcend speed and cost.
Published in Drug Discovery & Development magazine: Vol. 12, No. 3, March, 2009, pp.8-13.
Filed Under: Genomics/Proteomics