The proliferation of large-scale DNA-sequencing projects in recent years has driven a search for alternative methods to reduce time and cost. Here we describe a scalable, highly parallel sequencing system with raw throughput significantly greater than that of state-of-the-art capillary electrophoresis instruments. The apparatus uses a novel fibre-optic slide of individual wells and is able to sequence 25 million bases, at 99% or better accuracy, in one four-hour run. To achieve an approximately 100-fold increase in throughput over current Sanger sequencing technology, we have developed an emulsion method for DNA amplification and an instrument for sequencing by synthesis using a pyrosequencing protocol optimized for solid support and picolitre-scale volumes. Here we show the utility, throughput, accuracy and robustness of this system by shotgun sequencing and de novo assembly of the Mycoplasma genitalium genome with 96% coverage at 99.96% accuracy in one run of the machine.DNA sequencing has markedly changed the nature of biomedical research and medicine. Reductions in the cost, complexity and time required to sequence large amounts of DNA, including improvements in the ability to sequence bacterial and eukaryotic genomes, will have significant scientific, economic and cultural impact. Largescale sequencing projects, including whole-genome sequencing, have usually required the cloning of DNA fragments into bacterial vectors, amplification and purification of individual templates, followed by Sanger sequencing 1 using fluorescent chain-terminating nucleotide analogues 2 and either slab gel or capillary electrophoresis. Current estimates put the cost of sequencing a human genome between $10 million and $25 million 3 . Alternative sequencing methods have been described 4-8 ; however, no technology has displaced the use of bacterial vectors and Sanger sequencing as the main generators of sequence information.Here we describe an integrated system whose throughput routinely enables applications requiring millions of bases of sequence information, including whole-genome sequencing. Our focus has been on the co-development of an emulsion-based method 9-11 to isolate and amplify DNA fragments in vitro, and of a fabricated substrate and instrument that performs pyrophosphate-based sequencing (pyrosequencing 5,12 ) in picolitre-sized wells.In a typical run we generate over 25 million bases with a Phred quality score of 20 or better (predicted to have an accuracy of 99% or higher). Although this Phred 20 quality throughput is significantly higher than that of Sanger sequencing by capillary electrophoresis, it is currently at the cost of substantially shorter reads and lower average individual read accuracy. Sanger-based capillary electrophoresis sequencing systems produce up to 700 bases of sequence information from each of 96 DNA templates at an average read accuracy of 99.4% in 1 h, or 67,000 bases per hour, with substantially all of the bases having Phred 20 or better quality 23 . We further characterize the performance ...

We describe an mRNA profiling technique for determining differential gene expression that utilizes, but does not require, prior knowledge of gene sequences. This method permits high-throughput reproducible detection of most expressed sequences with a sensitivity of greater than 1 part in 100,000. Gene identification by database query of a restriction endonuclease fingerprint, confirmed by competitive PCR using gene-specific oligonucleotides, facilitates gene discovery by minimizing isolation procedures. This process, called GeneCalling, was validated by analysis of the gene expression profiles of normal and hypertrophic rat hearts following in vivo pressure overload.

We have micromachined a silicon-chip device that transports DNA with a Brownian ratchet that rectifies the Brownian motion of microscopic particles. Transport properties for a DNA 50-mer agree with theoretical predictions, and the DNA diffusion constant agrees with previous experiments. This type of micromachine could provide a generic pump or separation component for DNA or other charged species as part of a microscale lab-on-a-chip. A device with reduced feature size could produce a size-based separation of DNA molecules, with applications including the detection of single-nucleotide polymorphisms. The Human Genome Project aims to provide the complete sequence of the 3 billion base pairs of the human genome. While the dominant method for analyzing DNA fragments remains gel electrophoresis, new technologies that have the potential to increase the rate and decrease the cost of DNA sequencing and analysis, such as mass spectrometry and hybridization arrays, are critical to the project's success (1).Here we describe a method of DNA transport and separation based on a Brownian ratchet. As described originally by Smoluchowski (2) and noted by Feynman (3), a Brownian particle can undergo net transport on a potential energy surface that is externally driven to fluctuate between several distinct states. Brownian ratchets have attracted theoretical attention (4-12) because of their description of molecular motors (13-15) and their similarity with phenomena termed stochastic resonance and resonance activation (16,17).Brownian ratchets have been demonstrated to transport micrometer-to millimeter-sized particles in experiments with ratchets generated by dielectrophoresis (18), optical tweezers (19), and electrocapillary forces (20,21). Other devices based on entropic ratchets (22) or physical barriers (23, 24) have been proposed as well. More recently, a geometrical sieve device has been used to separate phospholipids (25).Despite these successes, the Brownian ratchet mechanism has not before proved capable of transporting DNA fragments in the size ranges applicable to DNA sequencing (Ͻ1000 nt) because the interactions used to establish the ratchet potential were too weak. In contrast to previous devices using polarization interactions to generate ratchets (18,19), the device that we have fabricated uses charge-charge interactions to generate the ratchet potential. As seen below, the charge-charge interactions have sufficient strength to establish ratchets that can trap small DNA fragments.The ratchet-like wells that trap DNA are generated by charging a series of patterned electrodes. When the electrodes are discharged, the traps vanish and the molecules undergo Brownian motion. Next the potential is reapplied, and the particles again collect in the traps. A spatial asymmetry in the shape of each ratchet-shaped well rectifies the Brownian motion and produces net transport as the on-off cycle is repeated. Each molecule's transport rate depends on its diffusion constant, allowing the possibility of size-based separations. We...

We have begun a long-term ecological research project to address questions about the impact of multiple disturbances on the species richness of communities and whether multiple disturbances are additive or interactive. A protected water catchment area was chosen, which is subjected to fires, sand mining and clearing, and for which detailed records are available. The study area, at Tomago (32°52'S, 151°45'E), has forest, woodland, shrubland and swamp on a sand substrate, with the vegetated dunes forming part of a coastal embayment. Forty-four sites were located in forested areas that had undergone disturbance by either fire, sand mining or clearing. Sites of each disturbance type were grouped into four age classes: less than 1 year since disturbance, nominally 1991; 5 years, nominally 1986; 11 years, nominally 1980; and 17 years, nominally 1974. A set of burned sites, with the time of the last fire matched to the times of the other disturbances, was used as the control response. In this paper we describe the study area and sites, then examine the effects of each single disturbance on vegetation structure. Canopy cover increased with time and type of disturbance, with 17 year old cleared or mined sites similar to the cover of 11 year old burned sites. In the first two years after disturbance, burned sites had significantly more understorey vegetation than cleared or mined sites, but by 5 years all three were similar. The data presented here show that regeneration of mined sites at Tomago is substantially slower than regeneration following disturbance by fire, with the regeneration of cleared sites intermediate but closer to mining than fire. After 17 years regeneration, cleared and sand mined sites had not returned to the vegetation structure of the pre-disturbance state. Understorey height and the amount of vegetation on cleared or mined sites have not achieved the levels in the original forest, although canopy cover did seem to have reached pre-disturbance levels. Current rehabilitation techniques are more sophisticated than those used 17 years ago and continued monitoring of sites currently being rehabilitated may show a faster return to pre-disturbance states. Having established the hierarchy and nature of the response to each single disturbance here, we are now in a position to investigate the impact of multiple disturbances.