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Initial sequencing and comparative analysis of the mouse genome Mouse Genome Sequencing Consortium* *A list of authors and their affiliations appears at the end of the paper ......................... ..................................................................................................................................................................................................... The sequence of the mouse genome is a key informational tool for understanding the contents of the human genome and a key experimental tool for biomedical research. Here, we report the results of an international collaboration to produce a high-quality draft sequence of the mouse genome. We also present an initial comparative analysis of the mouse and human genomes, describing some of the insights that can be gleaned from the two sequences. We discuss topics including the analysis of the evolutionary forces shaping the size, structure and sequence of the genomes; the conservation of large-scale synteny across most of the genomes; the much lower extent of sequence orthology covering less than half of the genomes; the proportions of the genomes under selection; the number of protein-coding genes; the expansion of gene families related to reproduction and immunity; the evolution of proteins; and the identification of intraspecies polymorphism. With the complete sequence of the human genome nearly in hand1,2, the next challenge is to extract the extraordinary trove of information encoded within its roughly 3 billion nucleotides. This information includes the blueprints for all RNAs and proteins, the regulatory elements that ensure proper expression of all genes, the structural elements that govern chromosome function, and the records of our evolutionary history. Some of these features can be recognized easily in the human sequence, but many are subtle and difficult to discern. One of the most powerful general approaches for unlocking the secrets of the human genome is comparative genomics, and one of the most powerful starting points for comparison is the laboratory mouse, Mus musculus. Metaphorically, comparative genomics allows one to read evolution’s laboratory notebook. In the roughly 75 million years since the divergence of the human and mouse lineages, the process of evolution has altered their genome sequences and caused them to diverge by nearly one substitution for every two nucleotides (see below) as well as by deletion and insertion. The divergence rate is low enough that one can still align orthologous sequences, but high enough so that one can recognize many functionally important elements by their greater degree of conservation. Studies of small genomic regions have demonstrated the power of such cross-species conservation to identify putative genes or regulatory elements3–12. Genome-wide analysis of sequence conservation holds the prospect of systematically revealing such information for all genes. Genomewide comparisons among organisms can also highlight key differences in the forces shaping their genomes, including differences in mutational and selective pressures13,14. Literally, comparative genomics allows one to link laboratory notebooks of clinical and basic researchers. With knowledge of both genomes, biomedical studies of human genes can be complemented by experimental manipulations of corresponding mouse genes to accelerate functional understanding. In this respect, the mouse is unsurpassed as a model system for probing mammalian biology and human disease15,16. Its unique advantages include a century of genetic studies, scores of inbred strains, hundreds of spontaneous mutations, practical techniques for random mutagenesis, and, importantly, directed engineering of the genome through transgenic, knockout and knockin techniques17–22. For these and other reasons, the Human Genome Project (HGP) recognized from its outset that the sequencing of the human genome needed to be followed as rapidly as possible by the sequencing of the mouse genome. In early 2001, the International Human Genome Sequencing Consortium reported a draft sequence covering about 90% of the euchromatic human genome, with about 35% in finished form1 . Since then, progress towards a complete human sequence has proceeded swiftly, with approximately 98% of the genome now available in draft form and about 95% in finished form. Here, we report the results of an international collaboration involving centres in the United States and the United Kingdom to produce a high-quality draft sequence of the mouse genome and a broad scientific network to analyse the data. The draft sequence was generated by assembling about sevenfold sequence coverage from female mice of the C57BL/6J strain (referred to below as B6). The assembly contains about 96% of the sequence of the euchromatic genome (excluding chromosome Y) in sequence contigs linked together into large units, usually larger than 50 megabases (Mb). With the availability of a draft sequence of the mouse genome, we have undertaken an initial comparative analysis to examine the similarities and differences between the human and mouse genomes. Some of the important points are listed below. †The mouse genome is about 14% smaller than the human genome (2.5 Gb compared with 2.9 Gb). The difference probably reflects a higher rate of deletion in the mouse lineage. † Over 90% of the mouse and human genomes can be partitioned into corresponding regions of conserved synteny, reflecting segments in which the gene order in the most recent common ancestor has been conserved in both species. † At the nucleotide level, approximately 40% of the human genome can be aligned to the mouse genome. These sequences seem to represent most of the orthologous sequences that remain in both lineages from the common ancestor, with the rest likely to have been deleted in one or both genomes. †The neutral substitution rate has been roughly half a nucleotide substitution per site since the divergence of the species, with about twice as many of these substitutions having occurred in the mouse compared with the human lineage. †By comparing the extent of genome-wide sequence conservation to the neutral rate, the proportion of small (50–100 bp) segments in the mammalian genome that is under (purifying) selection can be estimated to be about 5%. This proportion is much higher than can be explained by protein-coding sequences alone, implying that the genome contains many additional features (such as untranslated regions, regulatory elements, non-protein-coding genes, and chromosomal structural elements) under selection for biological function. †The mammalian genome is evolving in a non-uniform mannerclones or DNA sequences to specific locations in the mouse genome. Other resources included large collections of expressed-sequence tags (EST)40, a growing number of full-length complementary DNAs41,42 and excellent bacterial artificial chromosome (BAC) libraries43. The latter have been used for deriving large sets of BAC-end sequences37 and, as part of this collaboration, to generate a fingerprint-based physical map44. Furthermore, key mouse genome databases were developed at the Jackson (http://www.informatics. jax.org/), Harwell (http://www.har.mrc.ac.uk/) and RIKEN (http://genome.rtc.riken.go.jp/) laboratories to provide the community with access to this information. With these resources, it became straightforward (but not always easy) to perform positional cloning of classic single-gene mutations for visible, behavioural, immunological and other phenotypes. Many of these mutations provide important models of human disease, sometimes recapitulating human phenotypes with uncanny accuracy. It also became possible for the first time to begin dissecting polygenic traits by genetic mapping of quantitative trait loci (QTL) for such traits. Continuing advances fuelled a growing desire for a complete sequence of the mouse genome. The development of improved random mutagenesis protocols led to the establishment of largescale screens to identify interesting new mutants, increasing the need for more rapid positional cloning strategies. QTL mapping experiments succeeded in localizing more than 1,000 loci affecting physiological traits, creating demand for efficient techniques capable of trawling through large genomic regions to find the underlying genes. Furthermore, the ability to perform directed mutagenesis of the mouse germ line through homologous recombination made it possible to manipulate any gene given its DNA sequence, placing an increasing premium on sequence information. In all of these cases, it was clear that genome sequence information could markedly accelerate progress. Origin of the Mouse Genome Sequencing Consortium With the sequencing of the human genome well underway by 1999, a concerted effort to sequence the entire mouse genome was organized by a Mouse Genome Sequencing Consortium (MGSC). The MGSC originally consisted of three large sequencing centres— the Whitehead/Massachusetts Institute of Technology (MIT) Center for Genome Research, the Washington University Genome Sequencing Center, and the Wellcome Trust Sanger Institute— together with an international database, Ensembl, a joint project between the European Bioinformatics Institute and the Sanger Institute. In addition to the genome-wide efforts of the MGSC, other publicly funded groups have been contributing to the sequencing of the mouse genome in specific regions of biological interest. Together, the MGSC and these programmes have so far yielded clone-based draft sequence consisting of 1,859 Mb (74%, although there is redundancy) and finished sequence of 477 Mb (19%) of the mouse genome. Furthermore, Mural and colleagues45 recently reported a draft sequence of mouse chromosome 16 containing 87 Mb (3.5%). To analyse the data reported here, the MGSC was expanded to include the other publicly funded sequencing groups and a Mouse Genome Analysis Group consisting of scientists from 27 institutions in 6 countries. Generating the draft genome sequence Sequencing strategy Sanger and co-workers developed the strategy of random shotgun sequencing in the early 1980s, and it has remained the mainstay of genome sequencing over the ensuing two decades. The approach involves producing random sequence ‘reads’, generating a preliminary assembly on the basis of sequence overlaps, and then performing directed sequencing to obtain a ‘finished’ sequence with gaps closed and ambiguities resolved46. Ansorge and colleagues47 extended the technique by the use of ‘paired-end sequencing’, in which sequencing is performed from both ends of a cloned insert to obtain linking information, which is then used in sequence assembly. More recently, Myers and co-workers48, and others, have developed efficient algorithms for exploiting such linking information. A principal issue in the sequencing of large, complex genomes has been whether to perform shotgun sequencing on the entire genome at once (whole-genome shotgun, WGS) or to first break the genome into overlapping large-insert clones and to perform shotgun sequencing on these intermediates (hierarchical shotgun)46. The WGS technique has the advantage of simplicity and rapid early coverage; it readily works for simple genomes with few repeats, but there can be difficulties encountered with genomes that contain highly repetitive sequences (such as the human genome, which has near-perfect repeats spanning hundreds of kilobases). Hierarchical shotgun sequencing overcomes such difficulties by using local assembly, thus decreasing the number of repeat copies in each assembly and allowing comparison of large regions of overlaps between clones. Consequently, efforts to produce finished sequences of complex genomes have relied on either pure hierarchical shotgun sequencing (including those of Caenorhabditis elegans49, Arabidopsis thaliana49 and human1 ) or a combination of WGS and hierarchical shotgun sequencing (including those of Drosophila melanogaster50, human2 and rice51). The ultimate aim of the MGSC is to produce a finished, richly annotated sequence of the mouse genome to serve as a permanent reference for mammalian biology. In addition, we wished to produce a draft sequence as rapidly as possible to aid in the interpretation of the human genome sequence and to provide a useful intermediate resource to the research community. Accordingly, we adopted a hybrid strategy for sequencing the mouse genome. The strategy has four components: (1) production of a BAC-based physical map of the mouse genome by fingerprinting and sequencing the ends of clones of a BAC library44; (2) WGS sequencing to approximately sevenfold coverage and assembly to generate an initial draft genome sequence; (3) hierarchical shotgun sequencing of BAC clones covering the mouse genome combined with the WGS data to create a hybrid WGS-BAC assembly; and (4) production of a finished sequence by using the BAC clones as a template for directed finishing. This mixed strategy was designed to exploit the simpler organizational aspects of WGS assemblies in the initial phase, while still culminating in the complete high-quality sequence afforded by clone-based maps. We chose to sequence DNA from a single mouse strain, rather than from a mixture of strains45, to generate a solid reference foundation, reasoning that polymorphic variation in other strains could be added subsequently (see below). After extensive consultation with the scientific community52, the B6 strain was selected because of its principal role in mouse genetics, including its wellcharacterized phenotype and role as the background strain on which many important mutations arose. We elected to sequence a female mouse to obtain equal coverage of chromosome X and autosomes. Chromosome Y was thus omitted, but this chromosome is highly repetitive (the human chromosome Y has multiple duplicated regions exceeding 100 kb in size with 99.9% sequence identity53) and seemed an unwise target for the WGS approach. Instead, mouse chromosome Y is being sequenced by a purely clonebased (hierarchical shotgun) approach. Sequencing and assembly The genome assembly was based on a total of 41.4 million sequence reads derived from both ends of inserts (paired-end reads) of various clone types prepared from B6 female DNA. The inserts ranged in size from 2 to 200 kb (Table 1). The three large MGSC [Show More]

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