Increasingly powerful sequencing technologies are ushering in an era of personal genome sequences and raising the possibility of using such information to guide medical decisions. Genome resequencing also promises to accelerate the identification of disease-associated mutations. Roughly 98% of the human genome is composed of repeats and intergenic or non-protein-coding sequences. Thus, it is crucial to focus resequencing on high-value genomic regions. Protein-coding exons represent one such type of high-value target. We have developed a method of using flexible, high-density microarrays to capture any desired fraction of the human genome, in this case corresponding to more than 200,000 protein-coding exons. Depending on the precise protocol, up to 55-85% of the captured fragments are associated with targeted regions and up to 98% of intended exons can be recovered. This methodology provides an adaptable route toward rapid and efficient resequencing of any sizeable, non-repeat portion of the human genome.

We applied high-density microarrays to the enrichment of specific sequences from the human genome for high-throughput sequencing. After capture of 6,726 approximately 500-base 'exon' segments, and of 'locus-specific' regions ranging in size from 200 kb to 5 Mb, followed by sequencing on a 454 Life Sciences FLX sequencer, most sequence reads represented selection targets. These direct selection methods supersede multiplex PCR for the large-scale analysis of genomic regions.

We applied whole-genome resequencing of Escherichia coli to monitor the acquisition and fixation of mutations that conveyed a selective growth advantage during adaptation to a glycerol-based growth medium. We identified 13 different de novo mutations in five different E. coli strains and monitored their fixation over a 44-d period of adaptation. We obtained proof that the observed spontaneous mutations were responsible for improved fitness by creating single, double and triple site-directed mutants that had growth rates matching those of the evolved strains. The success of this new genome-scale approach indicates that real-time evolution studies will now be practical in a wide variety of contexts.Comparative genomics has been almost entirely focused on genomic changes over long periods of time, on the order of millions of years. A new microarray-based method of whole-genome resequencing called comparative genome sequencing (CGS) 1 now makes it cost efficient to monitor bacterial evolution comprehensively over short time periods as well. This capability is important because many microbial phenomena, such as the emergence of new pathogens and the acquisition of antibiotic resistance factors, can occur over relatively short time scales. Experimental evolution of bacteria and viruses 2,3 is a facile approach to study these topics. It has been used to test predictions of evolutionary theory 4 and to study parallel changes in populations evolved for 20,000 generations 5 , acquisition of antibiotic resistance 1 and in vitro symbiosis 6 . Our laboratory has used experimental evolution as a tool for metabolic engineering 7 and to study the recovery of strains with gene knockouts in central metabolic genes 8 . Nevertheless, much remains unknown about genome plasticity over short evolutionary timescales.It is a common mistake to think of bacteria as static; that is, to assume that a culture grown overnight is the same as it was the day before. It has been estimated that nearly 10% of the individual bacteria in a Salmonella enterica population carry large-scale genome rearrangements 9 , and in a suboptimal environment, selection can alter a population very rapidly. The 10-20 generations that occur in the process of growing a bacterial culture are sufficient to create a heterogeneous population, depending on the magnitude of the selective advantage of adaptive mutations. This problem is avoided by using strains of bacteria that are adapted to common laboratory media, but there are interesting cases where a seemingly straightforward growth medium poses a great challenge to a bacterium.An example is E. coli K-12 grown in minimal medium supplemented with glycerol as the carbon and energy source. Despite a complete pathway for glycerol catabolism, large variations in the growth rates of various strains have been noted 10 . Growth of the sequenced strain MG1655 has been observed to differ from computational predictions based on flux balance analysis 11 . Upon extended logarithmic growth in glycerol minimal medium, the growth rate...

PA-824 is a promising new compound for the treatment of tuberculosis that is currently undergoing human trials. Like its progenitors metronidazole and CGI-17341, PA-824 is a prodrug of the nitroimidazole class, requiring bioreductive activation of an aromatic nitro group to exert an antitubercular effect. We have confirmed that resistance to PA-824 (a nitroimidazo-oxazine) and CGI-17341 (a nitroimidazo-oxazole) is most commonly mediated by loss of a specific glucose-6-phosphate dehydrogenase (FGD1) or its deazaflavin cofactor F 420, which together provide electrons for the reductive activation of this class of molecules. Although FGD1 and F 420 are necessary for sensitivity to these compounds, they are not sufficient and require additional accessory proteins that directly interact with the nitroimidazole. To understand more proximal events in the reductive activation of PA-824, we examined mutants that were wild-type for both FGD1 and F 420 and found that, although these mutants had acquired high-level resistance to PA-824 (and another nitroimidazo-oxazine), they retained sensitivity to CGI-17341 (and a related nitroimidazo-oxazole). Microarray-based comparative genome sequencing of these mutants identified lesions in Rv3547, a conserved hypothetical protein with no known function. Complementation with intact Rv3547 fully restored sensitivity to nitroimidazo-oxazines and restored the ability of Mtb to metabolize PA-824. These results suggest that the sensitivity of Mtb to PA-824 and related compounds is mediated by a protein that is highly specific for subtle structural variations in these bicyclic nitroimidazoles.comparative genome sequencing ͉ F420 ͉ nitroimidazole