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 ...

Epidemiological studies of the naturally transformable bacterial pathogen Streptococcus pneumoniae have previously been confounded by high rates of recombination. Sequencing 240 isolates of the PMEN1 (Spain(23F)-1) multidrug-resistant lineage enabled base substitutions to be distinguished from polymorphisms arising through horizontal sequence transfer. More than 700 recombinations were detected, with genes encoding major antigens frequently affected. Among these were 10 capsule-switching events, one of which accompanied a population shift as vaccine-escape serotype 19A isolates emerged in the USA after the introduction of the conjugate polysaccharide vaccine. The evolution of resistance to fluoroquinolones, rifampicin, and macrolides was observed to occur on multiple occasions. This study details how genomic plasticity within lineages of recombinogenic bacteria can permit adaptation to clinical interventions over remarkably short time scales.

The development and spread of antibiotic resistance in bacteria is a universal threat to both humans and animals that is generally not preventable, but can nevertheless be controlled and must be tackled in the most effective ways possible. To explore how the problem of antibiotic resistance might best be addressed, a group of thirty scientists from academia and industry gathered at the Banbury Conference Centre in Cold Spring Harbor, New York, May 16-18, 2011. From these discussions emerged a priority list of steps that need to be taken to resolve this global crisis.

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The spread of multidrug-resistant Staphylococcus aureus (MRSA) strains in the clinical environment has begun to pose serious limits to treatment options. Yet virtually nothing is known about how resistance traits are acquired in vivo. Here, we apply the power of whole-genome sequencing to identify steps in the evolution of multidrug resistance in isogenic S. aureus isolates recovered periodically from the bloodstream of a patient undergoing chemotherapy with vancomycin and other antibiotics. After extensive therapy, the bacterium developed resistance, and treatment failed. Sequencing the first vancomycin susceptible isolate and the last vancomycin nonsusceptible isolate identified genome wide only 35 point mutations in 31 loci. These mutations appeared in a sequential order in isolates that were recovered at intermittent times during chemotherapy in parallel with increasing levels of resistance. The vancomycin nonsusceptible isolates also showed a 100-fold decrease in susceptibility to daptomycin, although this antibiotic was not used in the therapy. One of the mutated loci associated with decreasing vancomycin susceptibility (the vraR operon) was found to also carry mutations in six additional vancomycin nonsusceptible S. aureus isolates belonging to different genetic backgrounds and recovered from different geographic sites. As costs drop, whole-genome sequencing will become a useful tool in elucidating complex pathways of in vivo evolution in bacterial pathogens.