All for All

On February 12, 2001, an unprecedented collection of papers describing the initial sequencing and analysis of the human genome was published in Nature and Science (International Human Genome Sequencing Consortium 2001; Venter et al. 2001). Although much celebration and press attention had surrounded the earlier announcement in June 2000 of the coverage of the vast majority of the human genome sequence in working draft form, the publications in February 2001 carried with them the kind of satisfying scientific significance that laborers in the genome fields had longed for—the full description of the methods used to determine the letters of over 90% of the human instruction book, and a host of surprising revelations from the analysis of its contents. This brief essay represents a personal reflection on how we got here, and where we are going. The achievement of these landmarks, coming years ahead of the original schedule, was only possible because of the advances begun early in the preceding decade, reflecting the polyphonic set of interconnected goals that the planners of the Human Genome Project (HGP) wisely included as part of the original master plan. Science traditionally operates by the process of researchers standing on the shoulders of those who came before, and that has certainly been true for the HGP. Building detailed genetic and physical maps, developing better, cheaper, and faster technologies for handling DNA, and mapping and sequencing the more modest-sized genomes of model organisms were all critical stepping stones on the path to initiating the large-scale sequencing of the human genome. Pilot efforts to sequence the human genome began in the mid-1990s. When the International Human Genome Sequencing Consortium met for the first time in Bermuda in 1996, there was a sense of excitement, but the magnitude of the task at hand was sobering– throughput was too low, costs were too high, technology was still immature. Despite that anxiety, the assembled scientific leaders from several countries at that meeting endorsed the importance of high quality sequence, and made one of the most crucial decisions of the genome era–immediate data release. Led by John Sulston and BobWaterston, whohad adopted this same policy for the sequence of C. elegans, the assembled sequencing center directors unanimously adopted a statement that all assembled contigs greater than 1 or 2 kb would be placed in public databases within 24 hours. The argument was simple: The sequence would only benefit the public fully if it could be understood, and that required making it immediately available so that all the creative minds of the planet could work on it. The establishment of this principle was one of the defining moments of the HGP. Over the next three years the rate-limiting steps of large-scale sequencing began to yield to creative innovations. The genome centers implemented major improvements in library production, template preparation, and laboratory information management, so that less and less human intervention was required in the main production pipelines. The advent of capillary sequencing machines from Amersham and ABD provided a much-needed boost in efficiency. Much has also been made of the appearance of a commercial entity on the scene in May 1998 (Celera Genomics) as an additional nudge to the HGP. Whereas it is fair to say that the resulting sense of competition provided an additional incentive to the genome centers, it would be misguided to say that the HGP was previously operating in a relaxed fashion, or that the significant advances in throughput would otherwise not have happened. After all, most of those advances were born of previous accomplishments of the HGP itself. From my perspective, a major turning point occurred in Houston in February 1999. The largest NIHfunded centers (at the Whitehead Institute, Washington University, and Baylor College of Medicine) had just undergone rigorous peer review of their proposals to scale up sequencing throughput and were about to receive a significant increase in funding. The Sanger Centre in Hinxton (UK) and the Joint Genome Institute (Walnut Creek, CA) of the Department of Energy were also scaling up production rapidly. An experiment carried out the preceding summer had documented the high degree of utility of a “working draft” human genome sequence; the half-dozen labs that compared draft and finished sequences found that the draft could answer most of the scientific queries they posed (although it was harder to work with), and suggested that the majority of the HGP’s efforts might well be devoted to obtaining working-draft coverage of the genome as quickly as possible, as long as the commitment to finishing was not diminished. Accordingly, the sequencing plans for the NIH and DOE that were E-MAIL: fc23a@nih.gov; FAX (301) 402-0837. Article and publication are at http://www.genome.org/cgi/doi/10.1101/ gr.1898. Commentary

At the First (1996) and Second (1997) International Strategy Meetings on Human DNA Sequencing, the attendees discussed and strongly endorsed the concept that human genomic DNA sequence produced by large-scale DNA sequencing centers funded by the Human Genome Project should release data as rapidly as possible. In the case of unfinished data, it was suggested that sequence assemblies of 1–2 kb in size should be released within 24 hr of generation. Finished data should be submitted to the public sequence databases on a similarly rapid time scale. Most of the funding agencies engaged in supporting the Human Genome Project have adopted policies that reflect the importance of rapid dissemination of genomic sequence data. Similarly the nucleotide sequence databases have made provision for the acceptance of the unfinished data as well as, of course, finished sequence data. At the 1998 meeting, the question was raised about similar data dissemination practices for genomic sequence data from other organisms. The attendees unanimously agreed that this was also critical and adopted the following statement as individual scientists. ‘‘As extensive determination of the genomic DNA sequence of several organisms proceeds, it is increasingly clear that sequence information has enormous and immediate scientific value, even prior to its final assembly and completion. Delaying the release of either unfinished or finished genomic DNA sequence data serves no useful purpose and actually has the effect of slowing the progress of research. Therefore, the attendees at the Third International Strategy Meeting on Human Genome Sequencing (Bermuda, Feb. 27–28, 1998) agreed unanimously to support, as individual scientists, the view that all publicly funded large-scale DNA sequencing projects, regardless of the organism, should deposit data immediately into the public domain, following the same guidelines that have previously been adopted by this group for human genomic sequence [see D.R. Bentley, Science 274: 533–534 (1996); http:// hugo.gdb.org/bermuda.htm]. The scientists attending this meeting will continue to adhere to these principles and urge all other scientists and policymaking groups involved in large-scale sequencing to adopt them as well.’’ This statement does not yet reflect, nor is it binding on, the policy of any funding agency. The attendees at the Third International Strategy Meeting on Human Genome Sequencing were Mark Adams, David Bentley, Bruce Birren, Martin Bobrow, Elbert Branscomb, Graham Cameron, Anthony V. Carrano, Ellson Chen, Francis Collins, David Cox, Mike Davies, Pieter de Jong, Richard Durbin, Glen Evans, Asao Fujiyama, Richard Gibbs, Eric Green, Phil Green, Mark Guyer, LaDeana Hillier, Ursula Hurtenbach, Elke Jordan, Jill Kent, Fumihiko Kikuchi, Steven Koonin, Jane Lamerdin, Eric Lander, Chris Martin, Richard McCombie, John McPherson, Michael Morgan, Richard Myers, James Ostell, Ari Patrinos, Jane Peterson, Bruce Roe, Jane Rogers, Andre Rosenthal, Yoshiyuki Sakaki, Barbara Skene, Edwin Southern, Jilly Steward, John Sulston, Mark Vaudin, Jeffrey Wadsworth, Robert Waterson, James Watson, Jean Weissenbach, Richard Wilson, and MarieLaure Yaspo.

We have sequenced a genomic clone of the gene encoding the mouse mitochondrial DNA polymerase. The gene consists of 23 exons, which span approximately 13.2 kb, with exons ranging in size from 53 to 768 bp. All intron-exon boundaries conform to the GT-AG rule. By comparison with the human genomic sequence, we found remarkable conservation of the gene structure; the intron-exon borders are in almost identical locations for the 22 introns. The 5' upstream region contains approximately 300 bp of homology between the mouse and human sequences that presumably contain the promoter element. This region lacks any obvious TATA domain and is relatively GC rich, consistent with the housekeeping function of the mitochondrial DNA polymerase. Finally, within the 5' flanking region, both mouse and human genes have a region of 73 bp with high homology to the tRNA-Arg gene.

The progress of human and mouse genome sequencing programs presages the possibility of systematic cross-species comparison of the two genomes as a powerful tool for gene and regulatory element identification. As the opportunities to perform comparative sequence analysis emerge, it is important to develop parameters for such analyses and to examine the outcomes of cross-species comparison. Our analysis used gene prediction and a database search of 430 kb of genomic sequence covering the Bpa/Str region of the mouse X chromosome, and 745 kb of genomic sequence from the homologous human X chromosome region. We identified 11 genes in mouse and 13 genes and two pseudogenes in human. In addition, we compared the mouse and human sequences using pairwise alignment and searches for evolutionary conserved regions (ECRs) exceeding a defined threshold of sequence identity. This approach aided the identification of at least four further putative conserved genes in the region. Comparative sequencing revealed that this region is a mosaic in evolutionary terms, with considerably more rearrangement between the two species than realized previously from comparative mapping studies. Surprisingly, this region showed an extremely high LINE and low SINE content, low G+C content, and yet a relatively high gene density, in contrast to the low gene density usually associated with such regions.

The sequence of the mouse genome allows one to compare the conservation of synteny between the human and mouse genome and exploration of regions that might have been involved in major rearrangements during the evolution of these two species (evolutionary genome rearrangements). Recent segmental duplications (or duplicons) are paralogous DNA sequences with high sequence identity that account for about 3.5-5% of the human genome and have emerged during the past approximately 35 million years of evolution. These regions are susceptible to illegitimate recombination leading to rearrangements that result in genomic disorders or genomic mutations. A catalogue of several hundred segmental duplications potentially leading to genomic rearrangements has been reported. The authors and others have observed that some chromosome regions involved in genomic disorders are shuffled in orientation and order in the mouse genome and that regions flanked by segmental duplications are often polymorphic. We have compared the human and mouse genome sequences and demonstrate here that recent segmental duplications correlate with breaks of synteny between these two species. We also observed that nine primary regions involved in human genomic disorders show changes in the order or the orientation of mouse/human synteny segments, were often flanked by segmental duplications in the human sequence. We found that 53% of all evolutionary rearrangement breakpoints associate with segmental duplications, as compared with 18% expected in a random location of breaks along the chromosome (P<0.0001). Our data suggest that segmental duplications have participated in the recent evolution of the human genome, as driving forces for evolutionary rearrangements, chromosome structure polymorphisms and genomic disorders.

Radiation hybrid map, physical map, and low-pass genomic sequence of the canine prcd region on CFA9 and comparative mapping with the syntenic region on human chromosome 17

Five chimpanzee bacterial artificial chromosome (BAC) sequences (described in GenBank) have been compared with the best matching regions of the human genome sequence to assay the amount and kind of DNA divergence. The conclusion is the old saw that we share 98.5% of our DNA sequence with chimpanzee is probably in error. For this sample, a better estimate would be that 95% of the base pairs are exactly shared between chimpanzee and human DNA. In this sample of 779 kb, the divergence due to base substitution is 1.4%, and there is an additional 3.4% difference due to the presence of indels. The gaps in alignment are present in about equal amounts in the chimp and human sequences. They occur equally in repeated and nonrepeated sequences, as detected by REPEATMASKER (http://ftp.genome.washington.edu/RM/RepeatMasker.html).

In a report issued in January 1988, the National Research Council (NRC) Committee on the Mapping and Sequencing of the Human Genome, on which the present authors served, recommended a staged mapping and sequencing project with early emphases on physical mapping of human DNA, mapping and sequencing of the genomes of model organisms, and the development of sequencing technology (1). As the Committee's recommendations on physical mapping are beginning to be implemented on a substantial scale, it is timely to review these recommendations in the light of recent technical advances. In particular, the polymerase chain reaction (PCR) (2), a method that has only come into widespread use during the past 2 years, seems to us to offer a path toward a physical map that largely circumvents two problems that were prominent in the NRC Committee's discussions. One of these was the difficulty of merging mapping data gathered by diverse methods in different laboratories into a consensus physical map. The second was the logistics and expense of managing the huge collections of cloned segments on which the mapping data would depend almost absolutely.

In this chapter, we focus on an institution that represents the exclusivity of the International Human Genome Sequencing Consortium (IHGSC) while also contributing to the ‘funnelling effect’ that shaped it: the Sanger Institute. After situating its origins in the sequencing of the worm Caenorhabditis elegans, we portray the Sanger Institute as a driver in the formation of the IHGSC and as an advocate for the concentration of its membership into a few high-throughput sequencing centres. The intensification of this genome centre model, alongside competition with the corporate sequencing effort led by Celera Genomics, shaped the formation of the IHGSC. As a result, the large-scale production of sequence data became an increasingly differentiated and exclusive practice undertaken by its members. Conversely, the smaller laboratories that used this data in their own genetics research were sidelined from the determination of the human reference sequence.

Pituitary prolactin, like growth hormone (GH) and several other protein hormones, shows an episodic pattern of molecular evolution in which sustained bursts of rapid change contrast with long periods of slow evolution. A period of rapid change occurred in the evolution of prolactin in primates, leading to marked sequence differences between human prolactin and that of nonprimate mammals. We have defined this burst more precisely by sequencing the coding regions of prolactin genes for a prosimian, the slow loris (Nycticebus pygmaeus), and a New World monkey, the marmoset (Callithrix jacchus). Slow loris prolactin is very similar in sequence to pig prolactin, so the episode of rapid change occurred during primate evolution, after the separation of lines leading to prosimians and higher primates. Marmoset prolactin is similar in sequence to human prolactin, so the accelerated evolution occurred before divergence of New World monkeys and Old World monkeys/apes. The burst of change was confined largely to coding sequence (nonsynonymous sites) for mature prolactin and is not marked in other components of the gene sequence. This and the observations that (1) there was no apparent loss of function during the episode of rapid evolution, (2) the rate of evolution slowed toward the basal rate after this burst, and (3) the distribution of substitutions in the prolactin molecule is very uneven support the idea that this episode of rapid change was due to positive adaptive selection. In the slow loris and marmoset there is no evidence for duplication of the prolactin gene, and evidence from another New World monkey (Cebus albifrons) and from the chimpanzee and human genome sequences, suggests that this is the general position in primates, contrasting with the situation for GH genes. The chimpanzee prolactin sequence differs from that of human at two residues and comparison of human and chimpanzee prolactin gene sequences suggests that noncoding regions associated with regulating expression may be evolving differently from other noncoding regions.

Recent advances in sequencing technologies have initiated an era of personal genome sequences. To date, human genome sequences have been reported for individuals with ancestry in three distinct geographical regions: a Yoruba African, two individuals of northwest European origin, and a person from China. Here we provide a highly annotated, whole-genome sequence for a Korean individual, known as AK1. The genome of AK1 was determined by an exacting, combined approach that included whole-genome shotgun sequencing (27.8x coverage), targeted bacterial artificial chromosome sequencing, and high-resolution comparative genomic hybridization using custom microarrays featuring more than 24 million probes. Alignment to the NCBI reference, a composite of several ethnic clades, disclosed nearly 3.45 million single nucleotide polymorphisms (SNPs), including 10,162 non-synonymous SNPs, and 170,202 deletion or insertion polymorphisms (indels). SNP and indel densities were strongly correlated genome-wide. Applying very conservative criteria yielded highly reliable copy number variants for clinical considerations. Potential medical phenotypes were annotated for non-synonymous SNPs, coding domain indels, and structural variants. The integration of several human whole-genome sequences derived from several ethnic groups will assist in understanding genetic ancestry, migration patterns and population bottlenecks.

To investigate clinical condyloma, abnormal cervical cytologic findings, and evidence of human papillomavirus infections, 89 adolescent girls were examined. Cellular DNAs extracted from exfoliated cervical cells were examined for human papillomavirus genomic sequences by Southern transfer hybridization using 32P-labeled human papillomavirus DNA probes. Human papillomavirus sequences were detected in 12 (13%) young women, abnormal cytologic specimens in 21 (24%), and vulvar condylomas in 12 (13%). The human papillomavirus types identified included HPV-6/11 (four instances), which is known to be associated with benign lesions, and HPV-16, -18, and -31 (eight instances) which are considered to have oncogenic potential. Two young women were infected with both HPV-16 and -31. Human papillomavirus sequences were found in 48% of the young women with abnormal cytologic findings and in 3% of patients with normal cytologic findings (P less than .0001). Condylomatous changes in the cervical smear were associated with the presence of HPV-6/11 and mild dysplasia with the presence of HPV-16, -18, and -31. The presence of vulvar condylomas correlated with condylomatous changes in the cervical smear and with the recovery of HPV-6/11 from the cervical epithelium. The results indicate that the prevalence of human papillomavirus infections in this population is high and that a majority of the infections are with viruses associated with lower genital tract malignancies.

The recent publications in Nature and Science by the Human Genome Consortium and Celera Genomics, respectively, while being landmark achievements in themselves, have also given pause for thought. A definitive catalogue of human genes is still not available but the broad picture of how humans compare with lower organisms at the genomic level is becoming clearer. The full impact of these findings on the practice of medicine is hard to predict, but research being conducted now, in the early years of the 21st century, will form the basis of future advances in the diagnosis and treatment of disease. Exactly what this will entail is the subject of intense debate, but there are some common starting points that were discussed at this meeting in Munich. The main theme to emerge was the need to move beyond the human genome sequence towards an understanding of proteins and their interactions in complex biological pathways, thereby increasing opportunities for drug discovery through the identification of new targets. The majority of the talks were therefore devoted to the description of technological advances in the analysis of gene and protein expression (and interaction) and in the use of various methods of gene deletion in order to validate individual proteins as drug targets. Perhaps it will still be a few years before it will be possible to report on the application of genomic analyses to routine medical practice at the first point of care for patients but when that happens, the research efforts described here will have been worthwhile.

Andrew Pollack, Celera to Quit Selling Genome Information, New York Times, April 27, 2005. (Requires registration.) Excerpt: &amp;#34;Celera Genomics, which raced with the publicly financed Human Genome Project to decipher the human DNA sequence, has decided to abandon the business of selling genetic information.

Cytogenetic map for Anopheles nili: Application for population genetics and comparative physical mapping