Search

Eric D. Green, Edward M. Rubin and Maynard V. Olson speculate on the next forty years of the applications, from policing to data storage.

It is well known that average levels of population structure are higher on the X chromosome compared to autosomes in humans. However, there have been surprisingly few analyses on the spatial distribution of population structure along the X chromosome. With publicly available data from the HapMap Project and Perlegen Sciences, we show a strikingly punctuated pattern of X chromosome population structure. Specifically, 87% of X-linked HapMap SNPs within the top 1% of F(ST) values cluster into five distinct loci. The largest of these regions spans 5.4 Mb and contains 66% of the most highly differentiated HapMap SNPs on the X chromosome. We demonstrate that the extreme clustering of highly differentiated SNPs on the X chromosome is not an artifact of ascertainment bias, nor is it specific to the populations genotyped in the HapMap Project. Rather, additional analyses and resequencing data suggest that these five regions have been substrates of recent and strong adaptive evolution. Finally, we discuss the implications that patterns of X-linked population structure have on the evolutionary history of African populations.

Genetic variation among individual humans occurs on many different scales, ranging from gross alterations in the human karyotype to single nucleotide changes. Here we explore variation on an intermediate scale--particularly insertions, deletions and inversions affecting from a few thousand to a few million base pairs. We employed a clone-based method to interrogate this intermediate structural variation in eight individuals of diverse geographic ancestry. Our analysis provides a comprehensive overview of the normal pattern of structural variation present in these genomes, refining the location of 1,695 structural variants. We find that 50% were seen in more than one individual and that nearly half lay outside regions of the genome previously described as structurally variant. We discover 525 new insertion sequences that are not present in the human reference genome and show that many of these are variable in copy number between individuals. Complete sequencing of 261 structural variants reveals considerable locus complexity and provides insights into the different mutational processes that have shaped the human genome. These data provide the first high-resolution sequence map of human structural variation--a standard for genotyping platforms and a prelude to future individual genome sequencing projects.

Udgivelsesdato: 2006-Apr-27 After the completion of a draft human genome sequence, the International Human Genome Sequencing Consortium has proceeded to finish and annotate each of the 24 chromosomes comprising the human genome. Here we describe the sequencing and analysis of human chromosome 3, one of the largest human chromosomes. Chromosome 3 comprises just four contigs, one of which currently represents the longest unbroken stretch of finished DNA sequence known so far. The chromosome is remarkable in having the lowest rate of segmental duplication in the genome. It also includes a chemokine receptor gene cluster as well as numerous loci involved in multiple human cancers such as the gene encoding FHIT, which contains the most common constitutive fragile site in the genome, FRA3B. Using genomic sequence from chimpanzee and rhesus macaque, we were able to characterize the breakpoints defining a large pericentric inversion that occurred some time after the split of Homininae from Ponginae, and propose an evolutionary history of the inversion.

Pyoverdine is the primary siderophore of the gram-negative bacterium Pseudomonas aeruginosa . The pyoverdine region was recently identified as the most divergent locus alignable between strains in the P. aeruginosa genome. Here we report the nucleotide sequence and analysis of more than 50 kb in the pyoverdine region from nine strains of P. aeruginosa . There are three divergent sequence types in the pyoverdine region, which correspond to the three structural types of pyoverdine. The pyoverdine outer membrane receptor fpvA may be driving diversity at the locus: it is the most divergent alignable gene in the region, is the only gene that showed substantial intratype variation that did not appear to be generated by recombination, and shows evidence of positive selection. The hypothetical membrane protein PA2403 also shows evidence of positive selection; residues on one side of the membrane after protein folding are under positive selection. R′, previously identified as a type IV strain, is clearly derived from a type III strain via a 3.4-kb deletion which removes one amino acid from the pyoverdine side chain peptide. This deletion represents a natural modification of the product of a nonribosomal peptide synthetase enzyme, whose consequences are predictive from the DNA sequence. There is also linkage disequilibrium between the pyoverdine region and pvdY , a pyoverdine gene separated by 30 kb from the pyoverdine region. The pyoverdine region shows evidence of horizontal transfer; we propose that some alleles in the region were introduced from other soil bacteria and have been subsequently maintained by diversifying selection.

We define ‘genetic individuality’ as intraspecies variation that has substantial heritability and involves traits that are sufficiently common that they can be observed in any modest–sized sampling of individuals. We propose that genetic individuality is largely shaped by the combinatory shuffling of a modest number of genes, each of which exists as a family of functionally and structurally diverged alleles. Unequivocal examples of such allele families are found at the O–antigen–biosynthetic locus inPseudomonas aeruginosaand the human leucocyte antigen locus in humans. We examine characteristic features of these allele families and explore the possibility that genetic loci with similar characteristics can be recognized in a whole–genome scan of human genetic variation.

Pseudomonas aeruginosa strains from the chronic lung infections of cystic fibrosis (CF) patients are phenotypically and genotypically diverse. Using strain PAO1 whole genome DNA microarrays, we assessed the genomic variation in P. aeruginosa strains isolated from young children with CF (6 months to 8 years of age) as well as from the environment. Eighty-nine to 97% of the PAO1 open reading frames were detected in 20 strains by microarray analysis, while subsets of 38 gene islands were absent or divergent. No specific pattern of genome mosaicism defined strains associated with CF. Many mosaic regions were distinguished by their low G + C content; their inclusion of phage related or pyocin genes; or by their linkage to a vgr gene or a tRNA gene. Microarray and phenotypic analysis of sequential isolates from individual patients revealed two deletions of greater than 100 kbp formed during evolution in the lung. The gene loss in these sequential isolates raises the possibility that acquisition of pyomelanin production and loss of pyoverdin uptake each may be of adaptive significance. Further characterization of P. aeruginosa diversity within the airways of individual CF patients may reveal common adaptations, perhaps mediated by gene loss, that suggest new opportunities for therapy.

Human chromosome 7 has historically received prominent attention in the human genetics community, primarily related to the search for the cystic fibrosis gene and the frequent cytogenetic changes associated with various forms of cancer. Here we present more than 153 million base pairs representing 99.4% of the euchromatic sequence of chromosome 7, the first metacentric chromosome completed so far. The sequence has excellent concordance with previously established physical and genetic maps, and it exhibits an unusual amount of segmentally duplicated sequence (8.2%), with marked differences between the two arms. Our initial analyses have identified 1,150 protein-coding genes, 605 of which have been confirmed by complementary DNA sequences, and an additional 941 pseudogenes. Of genes confirmed by transcript sequences, some are polymorphic for mutations that disrupt the reading frame.

Pseudomonas aeruginosa is a ubiquitous environmental bacterium that is of increasing importance as an opportunistic human pathogen. P. aeruginosa infections are common in patients with compromised antibacterial defenses. A particular niche for infections that has been created by advances in pediatric care is the lungs of cystic fibrosis (CF) patients. As effective management of other clinical complications has been introduced and life expectancy has increased, chronic pulmonary infections with P. aeruginosa have emerged as the leading cause of morbidity and mortality in CF (11, 17). Despite steady improvements in treatment, CF pulmonary infections resist eradication with antibiotics and lead, over a period of many years, to irreversible tissue destruction (11). Available data suggest that most CF patients are infected by unique strains of P. aeruginosa acquired from environmental reservoirs (15, 30, 31) and that the infections are frequently clonal (5, 29, 36, 39). During the time that a particular strain is resident in the airways of a CF patient, considerable genetic adaptation occurs. A well-known example of this process is the conversion of strains from nonmucoid to mucoid phenotypes due to mutations in mucA (11, 21, 22), a gene whose product is a negative regulator of the biosynthesis of the secreted polysaccharide alginate. Another example is the frequent loss of O antigen (8, 14, 18, 28, 29), a change that also appears to be due to mutation. Restriction mapping of P. aeruginosa strains from diverse backgrounds indicates that a significant proportion of the variation among isolates is due to insertions and deletions of up to 500 kbp of genomic material (30, 34). In this respect, P. aeruginosa is superficially similar to Escherichia coli. Comparison of the pathogenic O157 and the nonpathogenic K-12 E. coli strains has revealed that much of their difference can be attributed to blocks of DNA that are strain specific (26). These so-called K islands and O islands comprise 12% of the genome of K-12 and 26% of the genome of O157, respectively, and are interspersed in a conserved backbone sequence with relatively little polymorphism (26). In P. aeruginosa, few strain-specific regions have been well characterized. One example is the 50-kbp P. aeruginosa genomic island 1, PAGI-1, found in many clinical isolates of P. aeruginosa in place of the 7-kbp of PAO1 sequence and hypothesized to play a role in evading the host immune response (20). Other examples include a 20-kbp island found in P. aeruginosa strain PAK, containing genes involved in glycosylation of a-type flagellin (2), and the recently described 11 groups of gene clusters at the O-antigen biosynthetic locus (27). However, despite the evidence indicating that genomic islands may play an important role in P. aeruginosa biology, little is known about the sequence and location of other islands. Sequence-based studies of genetic variation in P. aeruginosa appear to support the presence of conserved backbone sequences, similar to the E. coli model. Sequencing of six housekeeping genes in 19 environmental and clinical isolates revealed levels of genetic diversity even lower than in the E. coli comparisons, with average pairwise nucleotide substitution rates on the order of a few tenths of a percentage point (15). These data, along with those from restriction-mapping experiments, suggest that P. aeruginosa genomes have numerous strain-specific regions interspersed in a well-conserved backbone. More detailed studies of DNA sequence variation in P. aeruginosa are needed to define these backbone and strain-specific sequences, which may promote our understanding of the genetic determinants of pathogenicity in CF lung infections. The recent sequencing of the genome of the standard laboratory strain of P. aeruginosa, PAO1 (37), has laid the groundwork for more extensive studies of genomic variation in clinical and environmental isolates of this bacterium. We have carried out whole-genome-sample sequencing of two P. aeruginosa strains isolated from late-stage CF-related infections and a strain from an environmental source. These data provide a detailed view of the pattern of sequence variation among the three new strains relative to the PAO1 reference.

Temperate bacteriophages effect chromosomal evolution of their bacterial hosts, mediating rearrangements and the acquisition of novel genes from other taxa. Although the Haemophilus influenzae genome shows evidence of past phage-mediated lateral transfer, the phages presumed responsible have not been identified. To date, six different H. influenzae phages are known; of these, only the HP1/S2 group, which lyosogenizes exclusively Rd strains (which were originally encapsulated serotype d), is well characterized. Phages in this group are genetically very similar, with a highly conserved set of genes. Because the majority of H. influenzae strains are nonencapsulated (nontypeable), it is important to characterize phages infecting this larger, genetically more diverse group of respiratory pathogens. We have identified and sequenced HP2, a bacteriophage of nontypeable H. influenzae . Although related to the fully sequenced HP1 (and even more so to the partially sequenced S2) and similar in genetic organization, HP2 has a few novel genes and differs in host range; HP2 will not infect or lysogenize Rd strains. Genomic comparisons between HP1/S2 and HP2 suggest recent divergence, with new genes completely replacing old ones at certain loci. Sequence comparisons suggest that H. influenzae phages evolve by recombinational exchange of genes with each other, with cryptic prophages, and with the host chromosome.

The outer carbohydrate layer, or O antigen, of Pseudomonas aeruginosa varies markedly in different isolates of these bacteria, and at least 20 distinct O-antigen serotypes have been described. Previous studies have indicated that the major enzymes responsible for O-antigen synthesis are encoded in a cluster of genes that occupy a common genetic locus. We used targeted yeast recombinational cloning to isolate this locus from the 20 internationally recognized serotype strains. DNA sequencing of these isolated segments revealed that at least 11 highly divergent gene clusters occupy this region. Homology searches of the encoded protein products indicated that these gene clusters are likely to direct O-antigen biosynthesis. The O15 serotype strains lack functional gene clusters in the region analyzed, suggesting that O-antigen biosynthesis genes for this serotype are harbored in a different portion of the genome. The overall pattern underscores the plasticity of the P. aeruginosa genome, in which a specific site in a well-conserved genomic region can be occupied by any of numerous islands of functionally related DNA with diverse sequences.

The homologous recombination pathway in yeast is an ideal tool for the sequence-specific assembly of plasmids. Complementary 80-nucleotide oligonucleotides that overlap a vector and a target fragment were found to serve as efficient recombination linkers for fragment subcloning. Using electroporation, single-stranded 80-mers were adequate for routine plasmid construction. A cycloheximide-based counterselection was introduced to increase the specificity of cloning by homologous recombination relative to nonspecific vector background. Reconstruction experiments suggest this counterselection increased cloning specificity by 100-fold. Cycloheximide counterselection was used in conjunction with 80-bp linkers to subclone targeted regions from bacterial artificial chromosomes. This technology may find broad application in the final stages of completing the Human Genome Sequencing Project and in applications of BAC clones to the functional analysis of complex genomes.

Current models of protecting human subjects create a zero-sum game of privacy versus data utility. We propose shifting the paradigm to techniques that facilitate trust between researchers and participants., Fulfilling the promise of the genetic revolution requires the analysis of large datasets containing information from thousands to millions of participants. However, sharing human genomic data requires protecting subjects from potential harm. Current models rely on de-identification techniques in which privacy versus data utility becomes a zero-sum game. Instead, we propose the use of trust-enabling techniques to create a solution in which researchers and participants both win. To do so we introduce three principles that facilitate trust in genetic research and outline one possible framework built upon those principles. Our hope is that such trust-centric frameworks provide a sustainable solution that reconciles genetic privacy with data sharing and facilitates genetic research.

The 5.67-megabase genome of the plant pathogenAgrobacterium tumefaciensC58 consists of a circular chromosome, a linear chromosome, and two plasmids. Extensive orthology and nucleotide colinearity between the genomes ofA. tumefaciensand the plant symbiontSinorhizobium melilotisuggest a recent evolutionary divergence. Their similarities include metabolic, transport, and regulatory systems that promote survival in the highly competitive rhizosphere; differences are apparent in their genome structure and virulence gene complement. Availability of theA. tumefacienssequence will facilitate investigations into the molecular basis of pathogenesis and the evolutionary divergence of pathogenic and symbiotic lifestyles.

Pseudomonas aeruginosa , a ubiquitous gram-negative bacterium, is capable of colonizing a wide range of environmental niches and can also cause serious infections in humans. In order to understand the genetic makeup of pathogenic P. aeruginosa strains, a method of differential hybridization of arrayed libraries of cloned DNA fragments was developed. An M13 library of DNA from strain X24509, isolated from a patient with a urinary tract infection, was screened using a DNA probe from P. aeruginosa strain PAO1. The genome of PAO1 has been recently sequenced and can be used as a reference for comparisons of genetic organization in different strains. M13 clones that did not react with a DNA probe from PAO1 carried X24509-specific inserts. When a similar array hybridization analysis with DNA probes from different strains was used, a set of M13 clones which carried sequences present in the majority of human P. aeruginosa isolates from a wide range of clinical sources was identified. The inserts of these clones were used to identify cosmids encompassing a contiguous 48.9-kb region of the X24509 chromosome called PAGI-1 (for “ P. aeruginosa genomic island 1”). PAGI-1 is incorporated in the X24509 chromosome at a locus that shows a deletion of a 6,729-bp region present in strain PAO1. Survey of the incidence of PAGI-1 revealed that this island is present in 85% of the strains from clinical sources. Approximately half of the PAGI-1-carrying strains show the same deletion as X24509, while the remaining strains contain both the PAGI-1 sequences and the 6,729-bp PAO1 segment. Sequence analysis of PAGI-1 revealed that it contains 51 predicted open reading frames. Several of these genes encoded products with predictable function based on their sequence similarities to known genes, including insertion sequences, determinants of regulatory proteins, a number of dehydrogenase gene homologs, and two for proteins of implicated in detoxification of reactive oxygen species. It is very likely that PAGI-1 was acquired by a large number of P. aeruginosa isolates through horizontal gene transfer. The selection for its maintenance may be the consequence of expression of any one of the genes of unknown function or the genes which allow P. aeruginosa to survive under the conditions that generate reactive oxygen species. Alternatively, one or both of the transcriptional regulators encoded in PAGI-1 may control the expression of genes in the P. aeruginosa chromosome, which provides a selective advantage for strains that have acquired this genomic island.

Evolutionary change results from differences in the reproductive success of individuals with different genotypes. The downside of this process is easy to grasp: selection constantly purges deleterious mutations from the gene pool. However, we know remarkably little about evolution's upside—that is, about the types of mutations that commonly lead to increased fitness. To understand the biology of natural populations—including, most notably, that of the human—we need testable ideas about the types of mutations that evolution is likely to have favored in the recent past.

Multiple-complete-digest mapping is a DNA mapping technique based on complete-restriction-digest fingerprints of a set of clones that provides highly redundant coverage of the mapping target. The maps assembled from these fingerprints order both the clones and the restriction fragments. Maps are coordinated across three enzymes in the examples presented. Starting with yeast artificial chromosome contigs from the 7q31.3 and 7p14 regions of the human genome, we have produced cosmid-based maps spanning more than one million base pairs. Each yeast artificial chromosome is first subcloned into cosmids at a redundancy of ×15–30. Complete-digest fragments are electrophoresed on agarose gels, poststained, and imaged on a fluorescent scanner. Aberrant clones that are not representative of the underlying genome are rejected in the map construction process. Almost every restriction fragment is ordered, allowing selection of minimal tiling paths with clone-to-clone overlaps of only a few thousand base pairs. These maps demonstrate the practicality of applying the experimental and software-based steps in multiple-complete-digest mapping to a target of significant size and complexity. We present evidence that the maps are sufficiently accurate to validate both the clones selected for sequencing and the sequence assemblies obtained once these clones have been sequenced by a “shotgun” method.

The central preoccupation of human genetics is an effort to understand the genotypic basis of human phenotypic diversity. Although recent progress in identifying the genes that, when mutated, underlie major genetic diseases has been rapid, knowledge of the genetic influences on the vast range of variable, and at least partially heritable, traits that constitute the “normal” range of human phenotypic variation lags. Spectacular advances in our knowledge of human genetic variation have laid the groundwork for a synthesis of insights from medical genetics, population genetics, molecular evolution, and the study of human origins that places basic constraints on models of human genetic individuality. Balancing selection, local adaptation, mutation-selection balance, and founder effects have all extensively shaped contemporary genetic variation. Long-term-balancing selection appears largely to reflect the consequences of host-pathogen arms races. Local adaptation has been widespread—and involved responses to a plethora of selective pressures, some identifiable but most unknown. However, it appears to be a combination of mutation-selection balance and founder effects that largely accounts for genetic individuality. If true, this inference has major implications for future research programs in human genetics.

Clone-based genome maps can be constructed by determining the presence or absence of sequence-tagged sites (STSs) in a redundant collection of yeast artificial chromosome clones (YACs). While STS-content mapping has proven to be an effective means of ordering clone ends and STSs along chromosomes, the exact physical map positions of these landmarks are not determined. This fundamental weakness can be overcome by RecA-assisted restriction endonuclease (RARE) cleavage, a method that exploits the binding specificity on duplex DNA of a RecA-protein-oligodeoxynucleotide complex to enhance the cleavage specificity of a restriction endonuclease. This technique allows selective cleavage at individual members of a large set of restriction sites. RARE-cleavage mapping was applied to a contig comprising 5 overlapping YACs spanning 580 kb on human chromosome 14. An STS-content map comprising 10 YAC-end specific STSs and one internal STS was constructed. RARE cleavage was performed on 2 YACs that span the entire contig at the EcoRI sites defining the vector-insert junctions of all 5 YACs, as well as at a HhaI site within the STS that was initially used to screen the YAC library for the clones in the contig. The sizes of the RARE-cleavage fragments were measured by pulsed-field gel electrophoresis and used to convert the STS-content map into a true physical map that indicates precise positions of clone ends and STSs.

The genome sequence of the aceticlastic methanoarchaeon Methanosaeta concilii GP6, comprised of a 3,008,626-bp chromosome and an 18,019-bp episome, has been determined and exhibits considerable differences in gene content from that of Methanosaeta thermophila.

The Human Genome Project in the United States is now well underway. Its programmatic direction was largely set by a National Research Council report issued in 1988. The broad framework supplied by this report has survived almost unchanged despite an upheaval in the technology of genome analysis. This upheaval has primarily affected physical and genetic mapping, the two dominant activities in the present phase of the project. Advances in mapping techniques have allowed good progress toward the specific goals of the project and are also providing strong corollary benefits throughout biomedical research. Actual DNA sequencing of the genomes of the human and model organisms is still at an early stage. There has been little progress in the intrinsic efficiency of DNA-sequence determination. However, refinements in experimental protocols, instrumentation, and project management have made it practical to acquire sequence data on an enlarged scale. It is also increasingly apparent that DNA-sequence data provide a potent means of relating knowledge gained from the study of model organisms to human biology. There is as yet little indication that the infusion of technology from outside biology into the Human Genome Project has been effectively stimulated. Opportunities in this area remain large, posing substantial technical and policy challenges.

The public project's sequencing strategy involved producing a map of the human genome, and then pinning sequence to it. This helps to avoid errors in the sequence, especially in repetitive regions.

We have established strong linkage evidence that supports mapping autosomal-dominant sensory/motor neuropathy with ataxia (SMNA) to chromosome 7q22-q32. SMNA is a rare neurological disorder whose phenotype encompasses both the central and the peripheral nervous system. In order to identify a gene responsible for SMNA, we have undertaken a comprehensive genomic evaluation of the region of linkage, including evaluation for repeat expansion and small deletions or duplications, capillary sequencing of candidate genes, and massively parallel sequencing of all coding exons. We excluded repeat expansion and small deletions or duplications as causative, and through microarray-based hybrid capture and massively parallel short-read sequencing, we identified a nonsynonymous variant in the human interferon-related developmental regulator gene 1 (IFRD1) as a disease-causing candidate. Sequence conservation, animal models, and protein structure evaluation support the involvement of IFRD1 in SMNA. Mutation analysis of IFRD1 in additional patients with similar phenotypes is needed for demonstration of causality and further evaluation of its importance in neurological diseases.

Basic to the development of long-range physical maps of DNA are the detection and localization of landmarks within recombinant clones. Sequence-tagged sites (STSs), which are short stretches of DNA that can be specifically detected by the polymerase chain reaction (PCR), can be used as such landmarks. Our interest is to construct physical maps of whole human chromosomes by localizing STSs within yeast artificial chromosome (YAC) clones. Here we describe a generalized strategy for the systematic generation of large numbers of STSs specific for human chromosome 7. These STSs can be detected by PCR assays developed following the sequencing of anonymous pieces of chromosome 7 DNA, which was derived from flow-sorted chromosomes or from lambda clones made from DNA of a human-hamster hybrid cell line. Our approach for STS generation is tailored for the development of PCR assays capable of screening a large YAC library. In this study, we report the generation of 100 new STSs specific to human chromosome 7.

A physical map of the Saccharomyces cerevisiae genome is presented. It was derived by mapping the sites for two restriction endonucleases, SfiI and NotI, each of which recognizes an 8-bp sequence. DNA-DNA hybridization probes for genetically mapped genes and probes that span particular SfiI and NotI sites were used to construct a map that contains 131 physical landmarks--32 chromosome ends, 61 SfiI sites and 38 NotI sites. These landmarks are distributed throughout the non-rDNA component of the yeast genome, which comprises 12.5 Mbp of DNA. The physical map suggests that those genes that can be detected and mapped by standard genetic methods are distributed rather uniformly over the full physical extent of the yeast genome. The map has immediate applications to the mapping of genes for which single-copy DNA-DNA hybridization probes are available.

A 680-kb yeast artificial chromosome (YAC) that contains a functional copyof the human hypoxanthine phosphoribosyltransferase (HPRT) gene has been isolated. This YAC, yHPRT, and another YAC, yXY837, which contains the 3′ end of the HPRT gene, have been mapped with restriction enzymes that cleave human DNA infrequently. The HPRT gene lies near the center of yHPRT. Fusion of yHPRT-containing yeast spheroplasts with mouse L A-9 cells, which are HPRT-negative, gives rise to HPRT-positive colonies. These colonies contain the human HPRT gene and express human HPRT mRNA. Fusion of yeast with mammalian cells is an efficient way of testing the integrity and functionality of human DNA contained in YACs.

A strategy for using processed, digitized images of one-dimensional electrophoretic gels to facilitate the analysis of large sets of overlapping clones is described. The images are acquired from fluorescently stained gels or from transilluminated gel photographs using a cooled, solid-state charge-coupled device camera. By employing sets of bands in the size-standard lanes as reference points, all the gel images are spatially normalized to a common reference template. After normalization, lane images from different gels can be compared as though the gels had been electrophoresed resed under identical, uniform-field conditions. Applications of this procedure to the analysis of a large set of overlapping λ clones from chromosome VII of Saccharomyces cerevisiae and to the estimation of fragment sizes are illustrated.

We have developed an approach for screening ordered arrays of yeast artificial-chromosome (YAC) clones containing human DNA that is based on the polymerase chain reaction (PCR). This approach is designed to determine the locations of positive clones within a YAC library that is stored as individual clones in 96-well microtiter plates. The high sensitivity and specificity of the PCR allow the detection of target sequences in DNA prepared from pools of 1920 or more YAC clones. The PCR-based screening protocol is performed in two successive stages, which effectively limit the location of a positive clone to four microtiter plates (384 clones). Final localization of each positive clone is accomplished by conventional DNA.DNA hybridization using a single filter containing the YAC clones from the appropriate four microtiter plates. This PCR-based screening strategy has proven highly efficient, allowing the identification and isolation of numerous YAC clones containing specific human genes. The prospects of developing a strategy for screening YAC libraries based completely on PCR assays are discussed, as are the potential applications of this approach to the systematic analysis of the human genome.

The human genome sequence has been finished to very high standards; however, more than 340 gaps remained when the finished genome was published by the International Human Genome Sequencing Consortium in 2004. Using fosmid resources generated from multiple individuals, we targeted gaps in the euchromatic part of the human genome. Here we report 2,488,842 bp of previously unknown euchromatic sequence, 363,114 bp of which close 26 of 250 euchromatic gaps, or 10%, including two remaining euchromatic gaps on chromosome 19. Eight (30.7%) of the closed gaps were found to be polymorphic. These sequences allow complete annotation of several human genes as well as the assignment of mRNAs. The gap sequences are 2.3-fold enriched in segmentally duplicated sequences compared to the whole genome. Our analysis confirms that not all gaps within 'finished' genomes are recalcitrant to subcloning and suggests that the paired-end-sequenced fosmid libraries could prove to be a rich resource for completion of the human euchromatic genome.

Francisella tularensis is a bacterial pathogen that causes the zoonotic disease tularemia and is important to biodefense. Currently, the only vaccine known to confer protection against tularemia is a specific live vaccine strain (designated LVS) derived from a virulent isolate of Francisella tularensis subsp. holarctica . The origin and source of attenuation of this strain are not known. To assist with the design of a defined live vaccine strain, we sought to determine the genetic basis of the attenuation of LVS. This analysis relied primarily on the comparison between the genome of LVS and Francisella tularensis holarctica strain FSC200, which differ by only 0.08% of their nucleotide sequences. Under the assumption that the attenuation was due to a loss of function(s), only coding regions were examined in this comparison. To complement this analysis, the coding regions of two slightly more distantly related Francisella tularensis strains were also compared against the LVS coding regions. Thirty-five genes show unique sequence variations predicted to alter the protein sequence in LVS compared to the other Francisella tularensis strains. Due to these polymorphisms, the functions of 15 of these genes are very likely lost or impaired. Seven of these genes were demonstrated to be under stronger selective constraints, suggesting that they are the most probable to be the source of LVS attenuation and useful for a newly defined vaccine.

Fundamental biological knowledge and the technology to acquire it have been immeasurably advanced by past efforts to understand and manipulate the genomes of model organisms. Has the utility of bacteria, yeast, worms, flies, mice, plants, and other models now peaked and are humans poised to become the model organism of the future? The Genetics Society of America recently convened its 2006 meeting entitled “Genetic Analysis: Model Organisms to Human Biology” to examine the future role of genetic research. (Because of time limitations, the meeting was unable to cover the substantial contributions and future potential of research on model prokaryotic organisms.) In fact, the potential of model-organism-based studies has grown substantially in recent years. The genomics revolution has revealed an underlying unity between the cells and tissues of eukaryotic organisms from yeast to humans. No uniquely human biological mechanisms have yet come to light. This common evolutionary heritage makes it possible to use genetically tractable organisms to model important aspects of human medical disorders such as cancer, birth defects, neurological dysfunction, reproductive failure, malnutrition, and aging in systems amenable to rapid and powerful experimentation. Applying model systems in this way will allow us to identify common genes, proteins, and processes that underlie human medical conditions. It will allow us to systematically decipher the gene–gene and gene–environment interactions that influence complex multigenic disorders. Above all, disease models have the potential to address a growing gap between our ability to collect human genetic data and to productively interpret and apply it. If model organism research is supported with these goals in mind, we can look forward to diagnosing and treating human disease using information from multiple systems and to a medical science built on the unified history of life on earth.

Allelic variation in codons that specify amino acids that line the peptide-binding pockets of HLA's Class II antigen-presenting proteins is superimposed on strikingly few deeply diverged haplotypes. These haplotypes appear to have been evolving almost independently for tens of millions of years. By complete resequencing of 20 haplotypes across the ∼100-kbp region that spans theHLA-DQA1, -DQB1, and -DRB1genes, we provide a detailed view of the way in which the genome structure at this locus has been shaped by the interplay of selection, gene-gene interaction, and recombination.

Currently, challenges exist to acquire long-range (hundreds of kilobase pairs) phase-discriminated sequence across substantial numbers of individuals. We have developed a straightforward method for isolating and characterizing specific genomic regions in a haplospecific manner. Real-time PCR is carried out to STS content map and genotype pools of fosmid clones arrayed in 384-well microtiter plates. Single-nucleotide polymorphisms, microsatellite markers, and insertion-deletion polymorphisms are used to differentiate the target region into haplotype-specific tiling paths. DNA of clones from these tiling paths is retrieved from the library and either sequenced by standard shotgun methods or amplified in vitro and sequenced by a primer-based, directed method. This approach provides convenient access to complete, haplotype-resolved resequencing data from multiple individuals across tens to hundreds of thousands of basepairs. We illustrate its implementation with a detailed example of more than 400 kbp from the human CFTR region, across 15 individuals, and summarize our experience applying it to many other human loci.

Inversions, deletions and insertions are important mediators of disease and disease susceptibility. We systematically compared the human genome reference sequence with a second genome (represented by fosmid paired-end sequences) to detect intermediate-sized structural variants >8 kb in length. We identified 297 sites of structural variation: 139 insertions, 102 deletions and 56 inversion breakpoints. Using combined literature, sequence and experimental analyses, we validated 112 of the structural variants, including several that are of biomedical relevance. These data provide a fine-scale structural variation map of the human genome and the requisite sequence precision for subsequent genetic studies of human disease.

The genome sequence of the genetically tractable, mesophilic, hydrogenotrophic methanogen Methanococcus maripaludis contains 1,722 protein-coding genes in a single circular chromosome of 1,661,137 bp. Of the protein-coding genes (open reading frames [ORFs]), 44% were assigned a function, 48% were conserved but had unknown or uncertain functions, and 7.5% (129 ORFs) were unique to M. maripaludis . Of the unique ORFs, 27 were confirmed to encode proteins by the mass spectrometric identification of unique peptides. Genes for most known functions and pathways were identified. For example, a full complement of hydrogenases and methanogenesis enzymes was identified, including eight selenocysteine-containing proteins, with each being paralogous to a cysteine-containing counterpart. At least 59 proteins were predicted to contain iron-sulfur centers, including ferredoxins, polyferredoxins, and subunits of enzymes with various redox functions. Unusual features included the absence of a Cdc6 homolog, implying a variation in replication initiation, and the presence of a bacterial-like RNase HI as well as an RNase HII typical of the Archaea . The presence of alanine dehydrogenase and alanine racemase, which are uniquely present among the Archaea , explained the ability of the organism to use l - and d -alanine as nitrogen sources. Features that contrasted with the related organism Methanocaldococcus jannaschii included the absence of inteins, even though close homologs of most intein-containing proteins were encoded. Although two-thirds of the ORFs had their highest Blastp hits in Methanocaldococcus jannaschii , lateral gene transfer or gene loss has apparently resulted in genes, which are often clustered, with top Blastp hits in more distantly related groups.

Pseudomonas aeruginosa strains from the chronic lung infections of cystic fibrosis (CF) patients are phenotypically and genotypically diverse. Using strain PAO1 whole genome DNA microarrays, we assessed the genomic variation in P. aeruginosa strains isolated from young children with CF (6 months to 8 years of age) as well as from the environment. Eighty-nine to 97% of the PAO1 open reading frames were detected in 20 strains by microarray analysis, while subsets of 38 gene islands were absent or divergent. No specific pattern of genome mosaicism defined strains associated with CF. Many mosaic regions were distinguished by their low G + C content; their inclusion of phage related or pyocin genes; or by their linkage to a vgr gene or a tRNA gene. Microarray and phenotypic analysis of sequential isolates from individual patients revealed two deletions of greater than 100 kbp formed during evolution in the lung. The gene loss in these sequential isolates raises the possibility that acquisition of pyomelanin production and loss of pyoverdin uptake each may be of adaptive significance. Further characterization of P. aeruginosa diversity within the airways of individual CF patients may reveal common adaptations, perhaps mediated by gene loss, that suggest new opportunities for therapy.

A method and apparatus for gel electrophoresis which employs periodic inversion of the electric field essentially in one dimension, denoted as field-inversion gel electrophoresis (FIGE), results in net migration by using a longer time or higher voltage in one direction than in the opposite direction. FIGE permits separation of DNA or protein mixtures in size ranges not accessible to ordinary electrophoresis.

Large-scale sequencing of the chimpanzee genome is now imminent. Beyond the inherent fascination of comparing the sequence of the human genome with that of our closest living relative, this project is likely to yield tangible scientific benefits in two areas. First, the discovery of functionally important mutations that are specific to the human lineage offers a new path towards medical benefits. Second, chimpanzee–human comparisons are likely to yield molecular insights into how new biological characteristics evolve — findings that might be relevant throughout the tree of life.

The Human Genome Project was a natural culmination of one of the great scientific triumphs of the 20th century—the elucidation of the means by which biological organisms store, replicate, and process information. Specifically, the discovery at mid-century that biological information is stored as a linear, digital code led directly to the concept of a genome sequence. Technical advances in DNA analysis during the second half of the 20th century made the determination of genome sequences a practical goal. The achievement of this goal, at the end of the century, occurred during an extraordinary confluence of rapid scientific progress, rampant technological optimism, and exuberant entrepreneurial capitalism. The resultant strains on basic scientific values are exemplified by the public‐ private competition that arose during the sequencing of the human genome. While this competition accelerated initial availability of the genome sequence, it did so at considerable cost to the health of the interface between science and society. Analysis of this episode may reveal important lessons since the human future will continue to be shaped by the same forces that were at play during the endgame of the Human Genome Project. There are two stories of the Human Genome Project. One describes a century of scientific progress that began with the rediscovery of Mendel’s laws in 1900 and ended in a frenzy of genome sequencing. The other is a story about contemporary societal values—particularly, those that framed the project’s endgame and continue to shape public perceptions toward this defining event of our time. Both stories deserve attention. The first will help counter the illusion that the Human Genome Project was a sudden inspiration of the 1990s, while the second offers a sobering look at the forces that shape the current interface between science and society. We should pay attention to the ethic of this frontier since—like that of the American West—its influence will linger long after the present exuberance has faded. I will start with the scientific story of the first triumphant century of genetics. In this narrative, the Human Genome Project will emerge as a natural step in the scientific quest to understand one of nature’s deepest mysteries: How can a fertilized egg cell, an object too small to see with the unaided eye, contain all the information required to guide

Microtubule associated protein tau (MAPT) encodes the microtubule associated protein tau, the primary component of neurofibrillary tangles found in Alzheimer's disease and other neurodegenerative disorders. Mutations in the coding and intronic sequences of MAPT cause autosomal dominant frontotemporal dementia (FTDP-17). MAPT is also a candidate gene for progressive supranuclear palsy and hereditary dysphagic dementia. A human PAC (201 kb) and a mouse BAC (161 kb) containing the entire MAPT and Mtapt genes, respectively, were identified and sequenced. Comparative DNA sequence analysis revealed over 100 conserved non-repeat potential cis-acting regulatory sequences in or close to MAPT. Those islands with greater than 67% nucleotide identity range in size from 20 to greater than 1700 nucleotides. Over 90 single nucleotide polymorphisms were identified in MAPT that are candidate susceptibility alleles for neurodegenerative disease. The 5′ and 3′ flanking genes for MAPT are the corticotrophin-releasing factor receptor (CRFR) gene and KIAA1267, a gene of unknown function expressed in brain.

Pseudomonas aeruginosa is a ubiquitous environmental bacterium that is one of the top three causes of opportunistic human infections. A major factor in its prominence as a pathogen is its intrinsic resistance to antibiotics and disinfectants. Here we report the complete sequence of P. aeruginosa strain PAO1. At 6.3 million base pairs, this is the largest bacterial genome sequenced, and the sequence provides insights into the basis of the versatility and intrinsic drug resistance of P. aeruginosa. Consistent with its larger genome size and environmental adaptability, P. aeruginosa contains the highest proportion of regulatory genes observed for a bacterial genome and a large number of genes involved in the catabolism, transport and efflux of organic compounds as well as four potential chemotaxis systems. We propose that the size and complexity of the P. aeruginosa genome reflect an evolutionary adaptation permitting it to thrive in diverse environments and resist the effects of a variety of antimicrobial substances.

The application of new technology to sequence the genome of an individual yields few biological insights. Nonetheless, the feat heralds an era of 'personal genomics' based on cheap sequencing.

Methods relying on dense arrays of synthetic oligodeoxynucleotides to target specific subsets of the human genome may enable routine resequencing of all human exons or multi-megabase-pair chromosomal regions.

The Human Genome Project is lurching toward large-scale genomic sequencing. Although we find the arguments in favor of this path compelling, there remain sobering uncertainties about the cost of producing genomic sequence on a gigabase-pair scale, the rate at which the needed sequencing capacity can be developed, and the extent to which compromises will be required in the quality of the final product. Of course, it is these very uncertainties that make the sequencing of the human genome a scientific and managerial challenge worthy of the committed attention of many hard-working and talented people. Policy decisions are now being made that will greatly affect how this talent is deployed during the years ahead. We argue here, on both scientific and managerial grounds, that it is essential that the Human Genome Project adopt a ‘‘quality-first’’ credo. Scientific arguments for quality are rooted in a view of how the sequence will be used. The most common uses of reference sequences involve comparisons with other data. A vast number of sequences, derived from many sources— human and nonhuman—will be compared with the human reference sequence by future scientists. We should not prejudge either the comparison methods or the questions that the comparisons will address. Our goal should be to produce a reference sequence sufficiently good that comparisons made with it will rarely fail or produce misleading results because the reference sequence is inaccurate or incomplete. The sequence differences that are detected should, in nearly all cases, reflect real biological effects or limitations on future experimental or theoretical methods—not errors in the reference data. Viewed from this perspective, the commonly accepted base-pair accuracy goal of 0.9999 is a minimum quality target. Current data suggest that two randomly chosen human genomes are likely to show sequence similarity at the 0.999 level. Hence, even at a 10 error rate, on the order of 10% of the discrepancies with the reference sequence that will arise during intraspecies comparisons will be due to errors in the reference data. Each such artifactual discrepancy will lower the efficiency or diminish the discrimination power of future scientific inquiries. Base-pair accuracy is only one dimension of sequence quality. Indeed, with current technology it is the least problematic one. The issues of sequence contiguity, clone validation, and assembly checking are both more challenging and more crucial from the perspective of sequence users. Contiguity has proven particularly elusive. Highly fragmented sequence, such as virtually all of the human genomic sequence currently in GenBank, is difficult either to use or to validate. At a contiguity standard of 100 kb, which a substantial portion of current GenBank entries fail to meet, many human genes are not even in a single piece. At a 1-Mb standard, candidate regions associated with positional cloning projects typically still contain gaps. Gaps not only leave users of the data uncertain as to the precise genomic origins and biological content of particular sequences, but they also make the sequences difficult to validate. Problems of all types accumulate in data adjacent to gaps because the self-consistency tests on which sequence validation depends fail in these regions. Gap sizes are difficult to measure reliably and often prove larger than expected. For sequence segments appreciably below 1 Mb in size, the relative orientations of adjacent segments—and even the ordering of the segments—can be difficult to determine. Clone validation involves demonstrating that a particular recombinant DNA molecule is a faithful replica of the genome from which it was derived. At present, the only practical method for validating large-insert clones is to check their consistency with other overlapping clones from the same region. This process can be carried out at various resolutions, ranging from restrictionfragment mapping to complete sequencing. Clone aberrations that occur reproducibly in most or all clones cannot be detected by these methods. However, experience indicates that such aberrations are rare. A more serious problem is that the self-consistency tests are difficult to carry out rigorously. Because nearly every clone in current libraries has a unique pair of end points and a given clone can come from either of two human haplotypes, self-consistency checking is often confounded by mapping errors and haplotype differences. Assembly checking involves experimental tests to ensure that sequence assembly programs have correctly melded individual sequence tracts to form the composite sequence. The most widely used method of assembly checking is to compare restriction digests of a recombinant DNA molecule with the pattern of fragment sizes predicted from the assembled sequence. Although we suspect that undetected clone validation errors are more common than undetected assembly errors, some trends in this area bear watching. For example, the increasing reliance on shotgun sampling of relatively large clones (e.g., 150to 200kb BAC clones) both increases the likelihood of assembly errors and makes them more difficult to detect. As this brief survey indicates, there are significant technical challenges associated with assessing the quality of genomic sequence data. However, these challenges can and must be met. In addition to the scientific rationale for Corresponding author. E-MAIL phg@u.washington.edu; FAX (206) 6857344. Insight/Outlook

An approach to restriction-site mapping and contig building that uses fragment-size data from multiple complete digests of a set of clones that oversample a genomic region is presented. Maps containing both fragment-length data and clone-end data are maintained for each restriction enzyme. Synchronization between the maps for the different enzymes is achieved by requiring the clone-end maps for all enzymes to be compatible. Basic concepts that underlie multiple-complete-digest mapping - including the match/merge approach to map incorporation, extension vs. assimilation, ambiguity, and clone-end compatibility - are presented. An initial application of multiple-complete-digest mapping to real data on a set of cosmid clones suggests that this mapping method has exceptional power to produce accurate maps that are well suited to the needs of large-scale DNA-sequencing projects. 31 refs., 7 figs., 2 tabs.