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We present haplotype-resolved reference genomes and comparative analyses of six ape species, namely: chimpanzee, bonobo, gorilla, Bornean orangutan, Sumatran orangutan, and siamang. We achieve chromosome-level contiguity with unparalleled sequence accuracy (<1 error in 500,000 base pairs), completely sequencing 215 gapless chromosomes telomere-to-telomere. We resolve challenging regions, such as the major histocompatibility complex and immunoglobulin loci, providing more in-depth evolutionary insights. Comparative analyses, including human, allow us to investigate the evolution and diversity of regions previously uncharacterized or incompletely studied without bias from mapping to the human reference. This includes newly minted gene families within lineage-specific segmental duplications, centromeric DNA, acrocentric chromosomes, and subterminal heterochromatin. This resource should serve as a definitive baseline for all future evolutionary studies of humans and our closest living ape relatives., Competing Interests: COMPETING INTERESTS E.E.E. is a scientific advisory board (SAB) member of Variant Bio, Inc. C.T.W. is a co-founder/CSO of Clareo Biosciences, Inc. W.L. is a co-founder/CIO of Clareo Biosciences, Inc. The other authors declare no competing interests.

Robertsonian chromosomes are a type of variant chromosome found commonly in nature. Present in one in 800 humans, these chromosomes can underlie infertility, trisomies, and increased cancer incidence. Recognized cytogenetically for more than a century, their origins have remained mysterious. Recent advances in genomics allowed us to assemble three human Robertsonian chromosomes completely. We identify a common breakpoint and epigenetic changes in centromeres that provide insight into the formation and propagation of common Robertsonian translocations. Further investigation of the assembled genomes of chimpanzee and bonobo highlights the structural features of the human genome that uniquely enable the specific crossover event that creates these chromosomes. Resolving the structure and epigenetic features of human Robertsonian chromosomes at a molecular level paves the way to understanding how chromosomal structural variation occurs more generally, and how chromosomes evolve.

Apes possess two sex chromosomes-the male-specific Y chromosome and the X chromosome, which is present in both males and females. The Y chromosome is crucial for male reproduction, with deletions being linked to infertility 1 . The X chromosome is vital for reproduction and cognition 2 . Variation in mating patterns and brain function among apes suggests corresponding differences in their sex chromosomes. However, owing to their repetitive nature and incomplete reference assemblies, ape sex chromosomes have been challenging to study. Here, using the methodology developed for the telomere-to-telomere (T2T) human genome, we produced gapless assemblies of the X and Y chromosomes for five great apes (bonobo (Pan paniscus), chimpanzee (Pan troglodytes), western lowland gorilla (Gorilla gorilla gorilla), Bornean orangutan (Pongo pygmaeus) and Sumatran orangutan (Pongo abelii)) and a lesser ape (the siamang gibbon (Symphalangus syndactylus)), and untangled the intricacies of their evolution. Compared with the X chromosomes, the ape Y chromosomes vary greatly in size and have low alignability and high levels of structural rearrangements-owing to the accumulation of lineage-specific ampliconic regions, palindromes, transposable elements and satellites. Many Y chromosome genes expand in multi-copy families and some evolve under purifying selection. Thus, the Y chromosome exhibits dynamic evolution, whereas the X chromosome is more stable. Mapping short-read sequencing data to these assemblies revealed diversity and selection patterns on sex chromosomes of more than 100 individual great apes. These reference assemblies are expected to inform human evolution and conservation genetics of non-human apes, all of which are endangered species., (© 2024. The Author(s).)

Since its initial release in 2000, the human reference genome has covered only the euchromatic fraction of the genome, leaving important heterochromatic regions unfinished. Addressing the remaining 8% of the genome, the Telomere-to-Telomere (T2T) Consortium presents a complete 3.055 billion-base pair sequence of a human genome, T2T-CHM13, that includes gapless assemblies for all chromosomes except Y, corrects errors in the prior references, and introduces nearly 200 million base pairs of sequence containing 1956 gene predictions, 99 of which are predicted to be protein coding. The completed regions include all centromeric satellite arrays, recent segmental duplications, and the short arms of all five acrocentric chromosomes, unlocking these complex regions of the genome to variational and functional studies.

After two decades of improvements, the current human reference genome (GRCh38) is the most accurate and complete vertebrate genome ever produced. However, no single chromosome has been finished end to end, and hundreds of unresolved gaps persist 1,2 . Here we present a human genome assembly that surpasses the continuity of GRCh38 2 , along with a gapless, telomere-to-telomere assembly of a human chromosome. This was enabled by high-coverage, ultra-long-read nanopore sequencing of the complete hydatidiform mole CHM13 genome, combined with complementary technologies for quality improvement and validation. Focusing our efforts on the human X chromosome 3 , we reconstructed the centromeric satellite DNA array (approximately 3.1 Mb) and closed the 29 remaining gaps in the current reference, including new sequences from the human pseudoautosomal regions and from cancer-testis ampliconic gene families (CT-X and GAGE). These sequences will be integrated into future human reference genome releases. In addition, the complete chromosome X, combined with the ultra-long nanopore data, allowed us to map methylation patterns across complex tandem repeats and satellite arrays. Our results demonstrate that finishing the entire human genome is now within reach, and the data presented here will facilitate ongoing efforts to complete the other human chromosomes.

Public health officials have raised concerns that plasmid transfer between Enterobacteriaceae species may spread resistance to carbapenems, an antibiotic class of last resort, thereby rendering common health care-associated infections nearly impossible to treat. To determine the diversity of carbapenemase-encoding plasmids and assess their mobility among bacterial species, we performed comprehensive surveillance and genomic sequencing of carbapenem-resistant Enterobacteriaceae in the National Institutes of Health (NIH) Clinical Center patient population and hospital environment. We isolated a repertoire of carbapenemase-encoding Enterobacteriaceae, including multiple strains of Klebsiella pneumoniae, Klebsiella oxytoca, Escherichia coli, Enterobacter cloacae, Citrobacter freundii, and Pantoea species. Long-read genome sequencing with full end-to-end assembly revealed that these organisms carry the carbapenem resistance genes on a wide array of plasmids. K. pneumoniae and E. cloacae isolated simultaneously from a single patient harbored two different carbapenemase-encoding plasmids, indicating that plasmid transfer between organisms was unlikely within this patient. We did, however, find evidence of horizontal transfer of carbapenemase-encoding plasmids between K. pneumoniae, E. cloacae, and C. freundii in the hospital environment. Our data, including full plasmid identification, challenge assumptions about horizontal gene transfer events within patients and identify possible connections between patients and the hospital environment. In addition, we identified a new carbapenemase-encoding plasmid of potentially high clinical impact carried by K. pneumoniae, E. coli, E. cloacae, and Pantoea species, in unrelated patients and in the hospital environment., (Copyright © 2014, American Association for the Advancement of Science.)

Although a considerable proportion of serum lipids loci identified in European ancestry individuals (EA) replicate in African Americans (AA), interethnic differences in the distribution of serum lipids suggest that some genetic determinants differ by ethnicity. We conducted a comprehensive evaluation of five lipid candidate genes to identify variants with ethnicity-specific effects. We sequenced ABCA1, LCAT, LPL, PON1, and SERPINE1 in 48 AA individuals with extreme serum lipid concentrations (high HDLC/low TG or low HDLC/high TG). Identified variants were genotyped in the full population-based sample of AA (n = 1694) and tested for an association with serum lipids. rs328 (LPL) and correlated variants were associated with higher HDLC and lower TG. Interestingly, a stronger effect was observed on a "European" vs. "African" genetic background at this locus. To investigate this effect, we evaluated the region among West Africans (WA). For TG, the effect size among WA was the same in AA with only African local ancestry (2-3% lower TG), while the larger association among AA with local European ancestry matched previous reports in EA (10%). For HDLC, there was no association with rs328 in AA with only African local ancestry or in WA, while the association among AA with European local ancestry was much greater than what has been observed for EA (15 vs. ∼ 5 mg/dl), suggesting an interaction with an environmental or genetic factor that differs by ethnicity. Beyond this ancestry effect, the importance of African ancestry-focused, sequence-based work was also highlighted by serum lipid associations of variants that were in higher frequency (or present only) among those of African ancestry. By beginning our study with the sequence variation present in AA individuals, investigating local ancestry effects, and seeking replication in WA, we were able to comprehensively evaluate the role of a set of candidate genes in serum lipids in AA.

Background: While Staphylococcus epidermidis is commonly isolated from healthy human skin, it is also the most frequent cause of nosocomial infections on indwelling medical devices. Despite its importance, few genome sequences existed and the most frequent hospital-associated lineage, ST2, had not been fully sequenced., Results: We cultivated 71 commensal S. epidermidis isolates from 15 skin sites and compared them with 28 nosocomial isolates from venous catheters and blood cultures. We produced 21 commensal and 9 nosocomial draft genomes, and annotated and compared their gene content, phylogenetic relatedness and biochemical functions. The commensal strains had an open pan-genome with 80% core genes and 20% variable genes. The variable genome was characterized by an overabundance of transposable elements, transcription factors and transporters. Biochemical diversity, as assayed by antibiotic resistance and in vitro biofilm formation, demonstrated the varied phenotypic consequences of this genomic diversity. The nosocomial isolates exhibited both large-scale rearrangements and single-nucleotide variation. We showed that S. epidermidis genomes separate into two phylogenetic groups, one consisting only of commensals. The formate dehydrogenase gene, present only in commensals, is a discriminatory marker between the two groups., Conclusions: Commensal skin S. epidermidis have an open pan-genome and show considerable diversity between isolates, even when derived from a single individual or body site. For ST2, the most common nosocomial lineage, we detect variation between three independent isolates sequenced. Finally, phylogenetic analyses revealed a previously unrecognized group of S. epidermidis strains characterized by reduced virulence and formate dehydrogenase, which we propose as a clinical molecular marker.

Ciliary dysfunction leads to a broad range of overlapping phenotypes, collectively termed ciliopathies. This grouping is underscored by genetic overlap, where causal genes can also contribute modifier alleles to clinically distinct disorders. Here we show that mutations in TTC21B, which encodes the retrograde intraflagellar transport protein IFT139, cause both isolated nephronophthisis and syndromic Jeune asphyxiating thoracic dystrophy. Moreover, although resequencing of TTC21B in a large, clinically diverse ciliopathy cohort and matched controls showed a similar frequency of rare changes, in vivo and in vitro evaluations showed a significant enrichment of pathogenic alleles in cases (P < 0.003), suggesting that TTC21B contributes pathogenic alleles to ∼5% of ciliopathy cases. Our data illustrate how genetic lesions can be both causally associated with diverse ciliopathies and interact in trans with other disease-causing genes and highlight how saturated resequencing followed by functional analysis of all variants informs the genetic architecture of inherited disorders.

Background: The approaches for shotgun-based sequencing of vertebrate genomes are now well-established, and have resulted in the generation of numerous draft whole-genome sequence assemblies. In contrast, the process of refining those assemblies to improve contiguity and increase accuracy (known as 'sequence finishing') remains tedious, labor-intensive, and expensive. As a result, the vast majority of vertebrate genome sequences generated to date remain at a draft stage., Results: To date, our genome sequencing efforts have focused on comparative studies of targeted genomic regions, requiring sequence finishing of large blocks of orthologous sequence (average size 0.5-2 Mb) from various subsets of 75 vertebrates. This experience has provided a unique opportunity to compare the relative effort required to finish shotgun-generated genome sequence assemblies from different species, which we report here. Importantly, we found that the sequence assemblies generated for the same orthologous regions from various vertebrates show substantial variation with respect to misassemblies and, in particular, the frequency and characteristics of sequence gaps. As a consequence, the work required to finish different species' sequences varied greatly. Application of the same standardized methods for finishing provided a novel opportunity to "assay" characteristics of genome sequences among many vertebrate species. It is important to note that many of the problems we have encountered during sequence finishing reflect unique architectural features of a particular vertebrate's genome, which in some cases may have important functional and/or evolutionary implications. Finally, based on our analyses, we have been able to improve our procedures to overcome some of these problems and to increase the overall efficiency of the sequence-finishing process, although significant challenges still remain., Conclusion: Our findings have important implications for the eventual finishing of the draft whole-genome sequences that have now been generated for a large number of vertebrates.

Since its start, the Mammalian Gene Collection (MGC) has sought to provide at least one full-protein-coding sequence cDNA clone for every human and mouse gene with a RefSeq transcript, and at least 6200 rat genes. The MGC cloning effort initially relied on random expressed sequence tag screening of cDNA libraries. Here, we summarize our recent progress using directed RT-PCR cloning and DNA synthesis. The MGC now contains clones with the entire protein-coding sequence for 92% of human and 89% of mouse genes with curated RefSeq (NM-accession) transcripts, and for 97% of human and 96% of mouse genes with curated RefSeq transcripts that have one or more PubMed publications, in addition to clones for more than 6300 rat genes. These high-quality MGC clones and their sequences are accessible without restriction to researchers worldwide.

ClinSeq is a pilot project to investigate the use of whole-genome sequencing as a tool for clinical research. By piloting the acquisition of large amounts of DNA sequence data from individual human subjects, we are fostering the development of hypothesis-generating approaches for performing research in genomic medicine, including the exploration of issues related to the genetic architecture of disease, implementation of genomic technology, informed consent, disclosure of genetic information, and archiving, analyzing, and displaying sequence data. In the initial phase of ClinSeq, we are enrolling roughly 1000 participants; the evaluation of each includes obtaining a detailed family and medical history, as well as a clinical evaluation. The participants are being consented broadly for research on many traits and for whole-genome sequencing. Initially, Sanger-based sequencing of 300-400 genes thought to be relevant to atherosclerosis is being performed, with the resulting data analyzed for rare, high-penetrance variants associated with specific clinical traits. The participants are also being consented to allow the contact of family members for additional studies of sequence variants to explore their potential association with specific phenotypes. Here, we present the general considerations in designing ClinSeq, preliminary results based on the generation of an initial 826 Mb of sequence data, the findings for several genes that serve as positive controls for the project, and our views about the potential implications of ClinSeq. The early experiences with ClinSeq illustrate how large-scale medical sequencing can be a practical, productive, and critical component of research in genomic medicine.

Human skin is a large, heterogeneous organ that protects the body from pathogens while sustaining microorganisms that influence human health and disease. Our analysis of 16S ribosomal RNA gene sequences obtained from 20 distinct skin sites of healthy humans revealed that physiologically comparable sites harbor similar bacterial communities. The complexity and stability of the microbial community are dependent on the specific characteristics of the skin site. This topographical and temporal survey provides a baseline for studies that examine the role of bacterial communities in disease states and the microbial interdependencies required to maintain healthy skin.

The many layers and structures of the skin serve as elaborate hosts to microbes, including a diversity of commensal and pathogenic bacteria that contribute to both human health and disease. To determine the complexity and identity of the microbes inhabiting the skin, we sequenced bacterial 16S small-subunit ribosomal RNA genes isolated from the inner elbow of five healthy human subjects. This analysis revealed 113 operational taxonomic units (OTUs; "phylotypes") at the level of 97% similarity that belong to six bacterial divisions. To survey all depths of the skin, we sampled using three methods: swab, scrape, and punch biopsy. Proteobacteria dominated the skin microbiota at all depths of sampling. Interpersonal variation is approximately equal to intrapersonal variation when considering bacterial community membership and structure. Finally, we report strong similarities in the complexity and identity of mouse and human skin microbiota. This study of healthy human skin microbiota will serve to direct future research addressing the role of skin microbiota in health and disease, and metagenomic projects addressing the complex physiological interactions between the skin and the microbes that inhabit this environment.

It is understood that DNA and amino acid substitution rates are highly sequence context-dependent, e.g., C --> T substitutions in vertebrates may occur much more frequently at CpG sites and that cysteine substitution rates may depend on support of the context for participation in a disulfide bond. Furthermore, many applications rely on quantitative models of nucleotide or amino acid substitution, including phylogenetic inference and identification of amino acid sequence positions involved in functional specificity. We describe quantification of the context dependence of nucleotide substitution rates using baboon, chimpanzee, and human genomic sequence data generated by the NISC Comparative Sequencing Program. Relative mutation rates are reported for the 96 classes of mutations of the form 5' alphabetagamma 3' --> 5' alphadeltagamma 3', where alpha, beta, gamma, and delta are nucleotides and beta not equal delta, based on maximum likelihood calculations. Our results confirm that C --> T substitutions are enhanced at CpG sites compared with other transitions, relatively independent of the identity of the preceding nucleotide. While, as expected, transitions generally occur more frequently than transversions, we find that the most frequent transversions involve the C at CpG sites (CpG transversions) and that their rate is comparable to the rate of transitions at non-CpG sites. A four-class model of the rates of context-dependent evolution of primate DNA sequences, CpG transitions > non-CpG transitions approximately CpG transversions > non-CpG transversions, captures qualitative features of the mutation spectrum. We find that despite qualitative similarity of mutation rates among different genomic regions, there are statistically significant differences.

We report the generation and analysis of functional data from multiple, diverse experiments performed on a targeted 1% of the human genome as part of the pilot phase of the ENCODE Project. These data have been further integrated and augmented by a number of evolutionary and computational analyses. Together, our results advance the collective knowledge about human genome function in several major areas. First, our studies provide convincing evidence that the genome is pervasively transcribed, such that the majority of its bases can be found in primary transcripts, including non-protein-coding transcripts, and those that extensively overlap one another. Second, systematic examination of transcriptional regulation has yielded new understanding about transcription start sites, including their relationship to specific regulatory sequences and features of chromatin accessibility and histone modification. Third, a more sophisticated view of chromatin structure has emerged, including its inter-relationship with DNA replication and transcriptional regulation. Finally, integration of these new sources of information, in particular with respect to mammalian evolution based on inter- and intra-species sequence comparisons, has yielded new mechanistic and evolutionary insights concerning the functional landscape of the human genome. Together, these studies are defining a path for pursuit of a more comprehensive characterization of human genome function.

A key component of the ongoing ENCODE project involves rigorous comparative sequence analyses for the initially targeted 1% of the human genome. Here, we present orthologous sequence generation, alignment, and evolutionary constraint analyses of 23 mammalian species for all ENCODE targets. Alignments were generated using four different methods; comparisons of these methods reveal large-scale consistency but substantial differences in terms of small genomic rearrangements, sensitivity (sequence coverage), and specificity (alignment accuracy). We describe the quantitative and qualitative trade-offs concomitant with alignment method choice and the levels of technical error that need to be accounted for in applications that require multisequence alignments. Using the generated alignments, we identified constrained regions using three different methods. While the different constraint-detecting methods are in general agreement, there are important discrepancies relating to both the underlying alignments and the specific algorithms. However, by integrating the results across the alignments and constraint-detecting methods, we produced constraint annotations that were found to be robust based on multiple independent measures. Analyses of these annotations illustrate that most classes of experimentally annotated functional elements are enriched for constrained sequences; however, large portions of each class (with the exception of protein-coding sequences) do not overlap constrained regions. The latter elements might not be under primary sequence constraint, might not be constrained across all mammals, or might have expendable molecular functions. Conversely, 40% of the constrained sequences do not overlap any of the functional elements that have been experimentally identified. Together, these findings demonstrate and quantify how many genomic functional elements await basic molecular characterization.

Sequencing of full-insert clones from full-length cDNA libraries from both Xenopus laevis and Xenopus tropicalis has been ongoing as part of the Xenopus Gene Collection Initiative. Here we present 10,967 full ORF verified cDNA clones (8049 from X. laevis and 2918 from X. tropicalis) as a community resource. Because the genome of X. laevis, but not X. tropicalis, has undergone allotetraploidization, comparison of coding sequences from these two clawed (pipid) frogs provides a unique angle for exploring the molecular evolution of duplicate genes. Within our clone set, we have identified 445 gene trios, each comprised of an allotetraploidization-derived X. laevis gene pair and their shared X. tropicalis ortholog. Pairwise dN/dS, comparisons within trios show strong evidence for purifying selection acting on all three members. However, dN/dS ratios between X. laevis gene pairs are elevated relative to their X. tropicalis ortholog. This difference is highly significant and indicates an overall relaxation of selective pressures on duplicated gene pairs. We have found that the paralogs that have been lost since the tetraploidization event are enriched for several molecular functions, but have found no such enrichment in the extant paralogs. Approximately 14% of the paralogous pairs analyzed here also show differential expression indicative of subfunctionalization.

Identification of the specific cytogenetic abnormality is one of the critical steps for classification of acute myeloblastic leukemia (AML) which influences the selection of appropriate therapy and provides information about disease prognosis. However at present, the genetic complexity of AML is only partially understood. To obtain a comprehensive, unbiased, quantitative measure, we performed serial analysis of gene expression (SAGE) on CD15(+) myeloid progenitor cells from 22 AML patients who had four of the most common translocations, namely t(8;21), t(15;17), t(9;11), and inv(16). The quantitative data provide clear evidence that the major change in all these translocation-carrying leukemias is a decrease in expression of the majority of transcripts compared with normal CD15(+) cells. From a total of 1,247,535 SAGE tags, we identified 2,604 transcripts whose expression was significantly altered in these leukemias compared with normal myeloid progenitor cells. The gene ontology of the 1,110 transcripts that matched known genes revealed that each translocation had a uniquely altered profile in various functional categories including regulation of transcription, cell cycle, protein synthesis, and apoptosis. Our global analysis of gene expression of common translocations in AML can focus attention on the function of the genes with altered expression for future biological studies as well as highlight genes/pathways for more specifically targeted therapy.

Although the cost of generating draft-quality genomic sequence continues to decline, refining that sequence by the process of "sequence finishing" remains expensive. Near-perfect finished sequence is an appropriate goal for the human genome and a small set of reference genomes; however, such a high-quality product cannot be cost-justified for large numbers of additional genomes, at least for the foreseeable future. Here we describe the generation and quality of an intermediate grade of finished genomic sequence (termed comparative-grade finished sequence), which is tailored for use in multispecies sequence comparisons. Our analyses indicate that this sequence is very high quality (with the residual gaps and errors mostly falling within repetitive elements) and reflects 99% of the total sequence. Importantly, comparative-grade sequence finishing requires approximately 40-fold less reagents and approximately 10-fold less personnel effort compared to the generation of near-perfect finished sequence, such as that produced for the human genome. Although applied here to finishing sequence derived from individual bacterial artificial chromosome (BAC) clones, one could envision establishing routines for refining sequences emanating from whole-genome shotgun sequencing projects to a similar quality level. Our experience to date demonstrates that comparative-grade sequence finishing represents a practical and affordable option for sequence refinement en route to comparative analyses.

The National Institutes of Health's Mammalian Gene Collection (MGC) project was designed to generate and sequence a publicly accessible cDNA resource containing a complete open reading frame (ORF) for every human and mouse gene. The project initially used a random strategy to select clones from a large number of cDNA libraries from diverse tissues. Candidate clones were chosen based on 5'-EST sequences, and then fully sequenced to high accuracy and analyzed by algorithms developed for this project. Currently, more than 11,000 human and 10,000 mouse genes are represented in MGC by at least one clone with a full ORF. The random selection approach is now reaching a saturation point, and a transition to protocols targeted at the missing transcripts is now required to complete the mouse and human collections. Comparison of the sequence of the MGC clones to reference genome sequences reveals that most cDNA clones are of very high sequence quality, although it is likely that some cDNAs may carry missense variants as a consequence of experimental artifact, such as PCR, cloning, or reverse transcriptase errors. Recently, a rat cDNA component was added to the project, and ongoing frog (Xenopus) and zebrafish (Danio) cDNA projects were expanded to take advantage of the high-throughput MGC pipeline.

The laboratory rat (Rattus norvegicus) is an indispensable tool in experimental medicine and drug development, having made inestimable contributions to human health. We report here the genome sequence of the Brown Norway (BN) rat strain. The sequence represents a high-quality 'draft' covering over 90% of the genome. The BN rat sequence is the third complete mammalian genome to be deciphered, and three-way comparisons with the human and mouse genomes resolve details of mammalian evolution. This first comprehensive analysis includes genes and proteins and their relation to human disease, repeated sequences, comparative genome-wide studies of mammalian orthologous chromosomal regions and rearrangement breakpoints, reconstruction of ancestral karyotypes and the events leading to existing species, rates of variation, and lineage-specific and lineage-independent evolutionary events such as expansion of gene families, orthology relations and protein evolution.

Hembase (http://hembase.niddk.nih.gov) is an integrated browser and genome portal designed for web-based examination of the human erythroid transcriptome. To date, Hembase contains 15,752 entries from erythroblast Expressed Sequenced Tags (ESTs) and 380 referenced genes relevant for erythropoiesis. The database is organized to provide a cytogenetic band position, a unique name as well as a concise annotation for each entry. Search queries may be performed by name, keyword or cytogenetic location. Search results are linked to primary sequence data and three major human genome browsers for access to information considered current at the time of each search. Hembase provides interested scientists and clinical hematologists with a genome-based approach toward the study of erythroid biology.

The systematic comparison of genomic sequences from different organisms represents a central focus of contemporary genome analysis. Comparative analyses of vertebrate sequences can identify coding and conserved non-coding regions, including regulatory elements, and provide insight into the forces that have rendered modern-day genomes. As a complement to whole-genome sequencing efforts, we are sequencing and comparing targeted genomic regions in multiple, evolutionarily diverse vertebrates. Here we report the generation and analysis of over 12 megabases (Mb) of sequence from 12 species, all derived from the genomic region orthologous to a segment of about 1.8 Mb on human chromosome 7 containing ten genes, including the gene mutated in cystic fibrosis. These sequences show conservation reflecting both functional constraints and the neutral mutational events that shaped this genomic region. In particular, we identify substantial numbers of conserved non-coding segments beyond those previously identified experimentally, most of which are not detectable by pair-wise sequence comparisons alone. Analysis of transposable element insertions highlights the variation in genome dynamics among these species and confirms the placement of rodents as a sister group to the primates.

Duplications have long been postulated to be an important mechanism by which genomes evolve. Interspecies genomic comparisons are one method by which the origin and molecular mechanism of duplications can be inferred. By comparative mapping in human, mouse, and rat, we previously found evidence for a recent chromosome-fission event that occurred in the mouse lineage. Cytogenetic mapping revealed that the genomic segments flanking the fission site appeared to be duplicated, with copies residing near the centromere of multiple mouse chromosomes. Here we report the mapping and sequencing of the regions of mouse chromosomes 5 and 6 involved in this chromosome-fission event as well as the results of comparative sequence analysis with the orthologous human and rat genomic regions. Our data indicate that the duplications associated with mouse chromosomes 5 and 6 are recent and that the resulting duplicated segments share significant sequence similarity with a series of regions near the centromeres of the mouse chromosomes previously identified by cytogenetic mapping. We also identified pericentromeric duplicated segments shared between mouse chromosomes 5 and 1. Finally, novel mouse satellite sequences as well as putative chimeric transcripts were found to be associated with the duplicated segments. Together, these findings demonstrate that pericentromeric duplications are not restricted to primates and may be a common mechanism for genome evolution in mammals.

The National Institutes of Health Mammalian Gene Collection (MGC) Program is a multiinstitutional effort to identify and sequence a cDNA clone containing a complete ORF for each human and mouse gene. ESTs were generated from libraries enriched for full-length cDNAs and analyzed to identify candidate full-ORF clones, which then were sequenced to high accuracy. The MGC has currently sequenced and verified the full ORF for a nonredundant set of >9,000 human and >6,000 mouse genes. Candidate full-ORF clones for an additional 7,800 human and 3,500 mouse genes also have been identified. All MGC sequences and clones are available without restriction through public databases and clone distribution networks (see http:mgc.nci.nih.gov).

In parallel with the production of genomic sequence data, attention is being focused on the generation of comprehensive cDNA-sequence resources. Such efforts are increasingly emphasizing the production of high-accuracy sequence corresponding to the entire insert of cDNA clones, especially those presumed to reflect the full-length mRNA. The complete sequencing of cDNA clones on a large scale presents unique challenges because of the generally small, yet heterogeneous, sizes of the cloned inserts. We have developed a strategy for high-throughput sequencing of cDNA clones using the transposon Tn5. This approach has been tailored for implementation within an existing large-scale 'shotgun-style' sequencing program, although it could be readily adapted for use in virtually any sequencing environment. In addition, we have developed a modified version of our strategy that can be applied to cDNA clones with large cloning vectors, thereby overcoming a potential limitation of transposon-based approaches. Here we describe the details of our cDNA-sequencing pipeline, including a summary of the experience in sequencing more than 4200 cDNA clones to produce more than 8 million base pairs of high-accuracy cDNA sequence. These data provide both convincing evidence that the insertion of Tn5 into cDNA clones is sufficiently random for its effective use in large-scale cDNA sequencing as well as interesting insight about the sequence context preferred for insertion by Tn5.

Williams syndrome is a complex developmental disorder that results from the heterozygous deletion of a approximately 1.6-Mb segment of human chromosome 7q11.23. These deletions are mediated by large (approximately 300 kb) duplicated blocks of DNA of near-identical sequence. Previously, we showed that the orthologous region of the mouse genome is devoid of such duplicated segments. Here, we extend our studies to include the generation of approximately 3.3 Mb of genomic sequence from the mouse Williams syndrome region, of which just over 1.4 Mb is finished to high accuracy. Comparative analyses of the mouse and human sequences within and immediately flanking the interval commonly deleted in Williams syndrome have facilitated the identification of nine previously unreported genes, provided detailed sequence-based information regarding 30 genes residing in the region, and revealed a number of potentially interesting conserved noncoding sequences. Finally, to facilitate comparative sequence analysis, we implemented several enhancements to the program, including the addition of links from annotated features within a generated percent-identity plot to specific records in public databases. Taken together, the results reported here provide an important comparative sequence resource that should catalyze additional studies of Williams syndrome, including those that aim to characterize genes within the commonly deleted interval and to develop mouse models of the disorder.

The sequencing of expressed sequence tags (ESTs) from Xenopus laevis has lagged behind efforts on many other common experimental organisms and man, partly because of the pseudotetraploid nature of the Xenopus genome. Nonetheless, large collections of Xenopus ESTs would be useful in gene discovery, oligonucleotide-based knockout studies, gene chip analyses of normal and perturbed development, mapping studies in the related diploid frog X. tropicalis, and for other reasons. We have created a normalized library of cDNAs from unfertilized Xenopus eggs. These cells contain all of the information necessary for the first several cell divisions in the early embryo, as well as much of the information needed for embryonic pattern formation and cell fate determination. To date, we have successfully sequenced 13,879 ESTs out of 16,607 attempts (83.6% success rate), with an average sequence read length of 508 bp. Using a fragment assembly program, these ESTs were assembled into 8,985 'contigs' comprised of up to 11 ESTs each. When these contigs were used to search publicly available databases, 46.2% bore no relationship to protein or DNA sequences in the database at the significance level of 1e-6. Examination of a sample of 100 of the assembled contigs revealed that most ( approximately 87%) were comprised of two apparent allelic variants. Expression profiles of 16 of the most prominent contigs showed that 12 exhibited some degree of zygotic expression. These findings have implications for sequence-specific applications for Xenopus ESTs, particularly the use of allele-specific oligonucleotides for knockout studies, differential hybridization techniques such as gene chip analysis, and the establishment of accurate nomenclature and databases for this species.

Identification and annotation of all the genes in the sequenced Drosophila genome is a work in progress. Wild-type testis function requires many genes and is thus of potentially high value for the identification of transcription units. We therefore undertook a survey of the repertoire of genes expressed in the Drosophila testis by computational and microarray analysis. We generated 3141 high-quality testis expressed sequence tags (ESTs). Testis ESTs computationally collapsed into 1560 cDNA set used for further analysis. Of those, 11% correspond to named genes, and 33% provide biological evidence for a predicted gene. A surprising 47% fail to align with existing ESTs and 16% with predicted genes in the current genome release. EST frequency and microarray expression profiles indicate that the testis mRNA population is highly complex and shows an extended range of transcript abundance. Furthermore, >80% of the genes expressed in the testis showed onefold overexpression relative to ovaries, or gonadectomized flies. Additionally, >3% showed more than threefold overexpression at p <0.05. Surprisingly, 22% of the genes most highly overexpressed in testis match Drosophila genomic sequence, but not predicted genes. These data strongly support the idea that sequencing additional cDNA libraries from defined tissues, such as testis, will be important tools for refined annotation of the Drosophila genome. Additionally, these data suggest that the number of genes in Drosophila will significantly exceed the conservative estimate of 13,601.

McKusick-Kaufman syndrome (MKKS, MIM 236700) is a human developmental anomaly syndrome comprising hydrometrocolpos (HMC), postaxial polydactyly (PAP) and congenital heart disease (CHD). MKKS has been mapped in the Old Order Amish population to 20p12, between D20S162 and D20S894 (ref. 3). Here we describe the identification of a gene mutated in MKKS. We analysed the approximately 450-kb candidate region by sample sequencing, which revealed the presence of several known genes and EST clusters. We evaluated candidate transcripts by northern-blot analysis of adult and fetal tissues. We selected one transcript with widespread expression, MKKS, for analysis in a patient from the Amish pedigree and a sporadic, non-Amish case. The Old Order Amish patient was found to be homozygous for an allele that had two missense substitutions and the non-Amish patient was a compound heterozygote for a frameshift mutation predicting premature protein truncation and a distinct missense mutation. The MKKS predicted protein shows amino acid similarity to the chaperonin family of proteins, suggesting a role for protein processing in limb, cardiac and reproductive system development. We believe that this is the first description of a human disorder caused by mutations affecting a putative chaperonin molecule.

The identification of the cystic fibrosis transmembrane conductance regulator gene (CFTR) in 1989 represents a landmark accomplishment in human genetics. Since that time, there have been numerous advances in elucidating the function of the encoded protein and the physiological basis of cystic fibrosis. However, numerous areas of cystic fibrosis biology require additional investigation, some of which would be facilitated by information about the long-range sequence context of the CFTR gene. For example, the latter might provide clues about the sequence elements responsible for the temporal and spatial regulation of CFTR expression. We thus sought to establish the sequence of the chromosomal segments encompassing the human CFTR and mouse Cftr genes, with the hope of identifying conserved regions of biologic interest by sequence comparison. Bacterial clone-based physical maps of the relevant human and mouse genomic regions were constructed, and minimally overlapping sets of clones were selected and sequenced, eventually yielding approximately 1.6 Mb and approximately 358 kb of contiguous human and mouse sequence, respectively. These efforts have produced the complete sequence of the approximately 189-kb and approximately 152-kb segments containing the human CFTR and mouse Cftr genes, respectively, as well as significant amounts of flanking DNA. Analyses of the resulting data provide insights about the organization of the CFTR/Cftr genes and potential sequence elements regulating their expression. Furthermore, the generated sequence reveals the precise architecture of genes residing near CFTR/Cftr, including one known gene (WNT2/Wnt2) and two previously unknown genes that immediately flank CFTR/Cftr.

Nephropathic cystinosis is an autosomal recessive disorder caused by the defective transport of cystine out of lysosomes. Recently, the causative gene (CTNS) was identified and presumed to encode an integral membrane protein called cystinosin. Many of the disease-associated mutations in CTNS are deletions, including one >55 kb in size that represents the most common cystinosis allele encountered to date. In an effort to determine the precise genomic organization of CTNS and to gain sequence-based insight about the DNA within and flanking cystinosis-associated deletions, we mapped and sequenced the region of human chromosome 17p13 encompassing CTNS. Specifically, a bacterial artificial chromosome (BAC)-based physical map spanning CTNS was constructed by sequence-tagged site (STS)-content mapping. The resulting BAC contig provided the relative order of 43 STSs. Two overlapping BACs, which together contain all of the CTNS exons as well as extensive amounts of flanking DNA, were selected and subjected to shotgun sequencing. A total of 200,237 bp of contiguous, high-accuracy sequence was generated. Analysis of the resulting data revealed a number of interesting features about this genomic region, including the long-range organization of CTNS, insight about the breakpoints and intervening DNA associated with the common cystinosis-causing deletion, and structural information about five genes neighboring CTNS (human ortholog of rat vanilloid receptor subtype 1 gene, CARKL, TIP-1, P2X5, and HUMINAE). In particular, sequence analysis detected the presence of a novel gene (CARKL) residing within the most common cystinosis-causing deletion. This gene encodes a previously unknown protein that is predicted to function as a carbohydrate kinase. Interestingly, both CTNS and CARKL are absent in nearly half of all cystinosis patients (i.e., those homozygous for the common deletion). [The sequence data described in this paper have been submitted to the GenBank data library under accession nos. AF168787 and AF163573.]

A complete understanding of human erythropoiesis will require a robust description of transcriptional activity in hematopoietic cells that proliferate and differentiate in response to erythropoietin (EPO). For this purpose, we cultured peripheral blood mononuclear cells in the presence or in the absence of EPO and examined the transcriptional profile of those cells arising only in response to EPO. A distinct population of CD71( +) cells that demonstrated an average of six additional doublings in suspension culture and erythroid colony formation in methylcellulose was isolated. Suppression subtractive hybridization of mRNA isolated from those cells permitted the identification of transcribed genes. A summary of 719 expressed sequence tags (ESTs) describing 505 independent transcripts is provided here with a full analysis of each EST available at http://hembase.niddk.nih.gov. Several transcripts that matched genes previously reported in the context of erythroid differentiation including 4 cell surface proteins were expressed at this developmental stage. Active chromatin remodeling was suggested by the identification of 4 histone proteins, 4 high-mobility group proteins, 13 transcription factors, and 6 genes involved in DNA recombination and repair. Numerous genes associated with leukemic translocations were also recognized including topoisomerases I and II, nucleophosmin, Translin, EGR1, dek, pim-1, TFG, and MLL. In addition to known transcripts, 44 novel EST were discovered. This transcriptional profile provides the first genomic-scale description of gene activity in erythroid progenitor cells.

The construction of highly integrated and annotated physical maps of human chromosomes represents a critical goal of the ongoing Human Genome Project. Our laboratory has focused on developing a physical map of human chromosome 7, a approximately 170-Mb segment of DNA that corresponds to an estimated 5% of the human genome. Using a yeast artificial chromosome (YAC)-based sequence-tagged site (STS)-content mapping strategy, 2150 chromosome 7-specific STSs have been established and mapped to a collection of YACs highly enriched for chromosome 7 DNA. The STSs correspond to sequences generated from a variety of DNA sources, with particular emphasis placed on YAC insert ends, genetic markers, and genes. The YACs include a set of relatively nonchimeric clones from a human-hamster hybrid cell line as well as clones isolated from total genomic libraries. For map integration, we have localized 260 STSs corresponding to Genethon genetic markers and 259 STSs corresponding to markers orders by radiation hybrid (RH) mapping on our YAC contigs. Analysis of the data with the program SEGMAP results in the assembly of 22 contigs that are "anchored" on the Genethon genetic map, the RH map, and/or the cytogenetic map. These 22 contigs are ordered relative to one another, are (in all but 3 cases) oriented relative to the centromere and telomeres, and contain > 98% of the mapped STSs. The largest anchored YAC contig, accounting for most of 7p, contains 634 STSs and 1260 YACs. An additional 14 contigs, accounting for approximately 1.5% of the mapped STSs, are assembled but remain unanchored on either the genetic or RH map. Therefore, these 14 "orphan" contigs are not ordered relative to other contigs. In our contig maps, adjacent STSs are connected by two or more YACs in > 95% of cases. With 2150 mapped STSs, our map provides an average STS spacing of approximately 79 kb. The physical map we report here exceeds the goal of 100-kb average STS spacing and should provide an excellent framework for systematic sequencing of the chromosome.

The establishment and mapping of gene-specific DNA sequences greatly complement the ongoing efforts to map and sequence all human chromosomes. To facilitate our studies of human chromosome 7, we have generated and analyzed 2006 expressed-sequence tags (ESTs) derived from a collection of direct selection cDNA libraries that are highly enriched for human chromosome 7 gene sequences. Similarity searches indicate that approximately two-thirds of the ESTs are not represented by sequences in the public databases, including those in dbEST. In addition, a large fraction (68%) of the ESTs do not have redundant or overlapping sequences within our collection. Human DNA-specific sequence-tagged sites (STSs) have been developed from 190 of the ESTs. Remarkably, 180 (96%) of these STSs map to chromosome 7, demonstrating the robustness of chromosome enrichment in constructing the direct selection cDNA libraries. Thus far, 140 of these EST-specific STSs have been assigned unequivocally to YAC contigs that are distributed across the chromosome. Together, these studies provide > 2000 ESTs highly enriched for chromosome 7 gene sequences, 180 new chromosome 7 STSs corresponding to ESTs, and a definitive demonstration of the ability to enrich for chromosome-specific cDNAs by direct selection. Furthermore, the libraries, sequence data, and mapping information will contribute to the construction of a chromosome 7 transcript map.

Retinitis pigmentosa is a genetically heterogeneous disease that has autosomal dominant, autosomal recessive and X-linked forms. Autosomal dominant retinitis pigmentosa (adRP) has thus far been associated with eight distinct loci, including the rhodopsin and peripherin/RDS genes as well as unidentified genes on chromosomes 7p, 7q, 8q, 17p, 17q, and 19q. The RP10 locus for adRP on chromosome 7q was first mapped in a Spanish family; later, an unrelated American family was identified that also showed linkage to 7q. By combining the linkage results from both families, we are able to assign the disease gene to a 5-cM interval on 7q. Based on extensive physical mapping of this region, the genetic interval is now fully contained within a approximately 5-Mb segment on a well-defined YAC contig. These studies significantly reduce the size of the RP10 critical region, exclude a number of possible candidate genes, and provide the necessary cloned DNA for the positional cloning of the RP10 gene.

A new gene (galM) has been identified as the fourth cistron of the gal operon, encoding enzymes for the metabolism of galactose and lactose in Escherichia coli. Induction of the gal operon either from the gal promoters or from a neighboring prophage lambda promoter expresses the galM gene as well. The new structure of the gal operon from the promoter end is galE-galT-galK-galM in counter-clockwise orientation on the chromosome. Genetic and biochemical analyses have revealed that the galM gene product has mutarotase activity, which converts alpha-aldose to the beta-anomer. Unlike mutarotase from other bacteria in which the enzyme is primarily processed for export and secretion, the mutarotase from E. coli does not appear to be processed and yet is still found in periplasm (and culture media when overexpressed) in significant amounts. Although the interconversion of the sugar anomers occurs spontaneously in pure water in vitro, the in vivo formation of alpha-D-galactopyranose (the substrate for phosphorylation) from beta-D-galactopyranose (generated by beta-galactosidase hydrolysis of lactose) is largely dependent upon the presence of the mutarotase. This shows that efficient lactose metabolism requires mutarotase. These results give credence to the idea that the activity of intracellular water is not high enough to permit a simple extrapolation of observed in vitro reactions to in vivo situations in every case.