by Caitlin Smith
Not long ago, science undergraduates only read about the building blocks of DNA in introductory textbooks. These days it is unsurprising for undergrads to be trained in the use of high-tech automatic sequencers. Previously, these students and other researchers had used what is known as the Sanger method (after its originator, Frederick Sanger), which first emerged in the late 1970s, and is still the gold standard for work requiring long reads of up to one thousand bases with high accuracy. Even today, the Sanger method is often the best choice for de novo sequencing, resequencing, genotyping, and fragment analysis.
But the Sanger method has a few significant drawbacks for those who are trying to sequence large numbers of bases: it uses a lot of time, money, and lab infrastructure. To address these drawbacks, researchers and policy makers have been trying to actualize the idea of “next generation sequencing.” This term can refer simply to any new sequencing method that is not based on the Sanger method; it often additionally refers to technology to enable the “$1000 genome.” Many companies and researchers are chasing this Holy Grail: a method for cheap, fast, whole-genome sequencing. “DNA sequencing has become more of a commodity, and the technology development has moved towards the goal of the $1000 genome for clinical types of applications, [such as] clinical sequencing for point of care medicine and preventive medicine,” says Jeff Harford, LI-COR’s product marketing manager. We aren't there yet, but it won't be long. Here are some developments that are driving us towards the goal.
Longer reads with higher accuracy
For next-generation throughput combined with the need for longer reads, LI-COR can procure read lengths comparable to or exceeding those seen with the Sanger method, by using both their sequencing instrumentation (the 4300L DNA Analyzer) and reagents, such as their IRDye infrared dye labeled oligos and termination mixes. “Our infrared dyes provide high sensitivity and very long read lengths on the 4300L (e.g. 1200 bp),” says Harford. “This has been the unique sequencing niche addressed by LI-COR for sequencing technology.” The LI-COR sequencing system gives 99% accuracy for 1000 base reads.
Shorter reads with higher accuracy
The advantage of shorter read lengths is the ultra-high throughput pace you can achieve with increased parallelization. The sequencing company 454 Life Sciences, recently acquired by Roche Applied Science, has a next generation sequencing technology in its Genome Sequencer FLX that gives accuracy rivaling the traditional Sanger method. It also reads 20 million base pairs in 4.5 hours—a speed that is enabled by their PicoTiterPlate Device, a fiber optic plate that sends chemiluminescent signals from the sequencing reaction to the CCD camera, to be recorded in the instrument. Read lengths are 200–300 bases, with greater than 99.5% accuracy. The shorter read lengths of this system make it more suited to sequencing bacterial genomes, for example; de novo sequencing of large mammalian genomes requires longer read lengths to avoid redundancy.
Applied Biosystems’ new SOLiD™ system also relies on massive parallelization and shorter read lengths to achieve ultra-high throughput sequencing. The SOLiD™ system uses a proprietary technology called stepwise ligation. To ensure high accuracy, the system has 2-base encoding, another proprietary mechanism that interrogates each base twice for errors during sequencing. The SOLiD™ system can generate more than one gigabase of useable data per run—the equivalent of one-third of the human genome. The SOLiD™ systems will be available in October.
Nanopores and more
A method under development at Agilent, as well as in several academic labs, is the idea of nanopore sequencing, wherein a single molecule of DNA could be sequenced with no need for amplification. In principle, a single-stranded DNA molecule can be driven through a single pore or channel so tiny that it partially blocks the pore as it moves through. The pore’s electrical properties are altered by the block—in different ways, depending on the particular base that is blocking the pore at a given moment. This should, theoretically, allow a reading of each base as it slides through the nanopore, in a single-file fashion.
One problem with this idea is that the bases of the DNA molecules whiz through the nanopore too quickly to make accurate measurements of them. Sandip Ghosal, associate professor of mechanical engineering at Northwestern University, recently published a paper in which he describes the force that works to determine the speed of the bases as they move through the nanopore. This description, in the form of an equation based on classical hydrodynamics, may help scientists to slow the DNA’s movement through the pore—at least enough to tell one base from another. “The connection with practical application arises because the equation suggests a method for slowing down the translocation speed by fine tuning the zeta potential on the channel wall to match the corresponding value at the DNA surface,” says Ghosal. “A variety of methods exists to control or alter the surface zeta potential. There are various ways in which one may go about it—[for example] trying other materials for nanopores, using coatings, treating surfaces with ion beams, or switching buffers.”
Suite sequencing tools
For those looking for help with a variety of sequencing challenges, MWG offers a suite of services that combines the strengths of Sanger sequencing with the high-throughput advantage of next generation sequencing. MWG recently upgraded to the Roche Genome Sequencer FLX system for the latter type of service. MWG offers Sanger sequencing on ABI equipment with sequencing-by-synthesis technology and bioinformatics, allowing them to sequence and compare bacterial genomes in about 48 hours. Jutta Huber, head of global business development, explains that they offer “coverage sequencing or complete sequencing of unknown bacterial or fungal genomes, or sequencing of metagenomes. After generation of the draft data with the latest Genome Sequencer FLX technology, we offer scaffolding of contigs and gap closing based on Sanger technology. Comparative sequencing in combination with our bioinformatic services allows you to compare your strain of interest with already known genomes.”
The FLX system improves over its predecessor, the Genome Sequencer 20, by about 5-fold in throughput: it can sequence 100 million base pairs in 7.5 hours, with greater accuracy. “With the GS FLX, we can generate about 100 MB per run,” says Huber. Among the many services offered by MWG is “sequencing of PCR amplicons with the Genome Sequencer FLX,” explains Huber. “We help customers in the design of the amplicons or offer it as a service—useful for the detection of rare mutations and study of methylation patterns.”
Huber believes that an obstacle to faster progress will be “effective bioinformatic tools to handle the high amount of data generated by next generation sequencing technology systems.” In fact, Applied Biosystems recently announced an initiative to support researchers in the development of bioinformatics applications for next-generation DNA sequencing platforms. The initiative is meant to help scientists realize the potential of next-generation sequencing, and its application to medical delivery and discovery.
