The mouse (Mus musculus) is the premier animal model for understanding human disease and development. Here we show that a comprehensive understanding of mouse biology is only possible with the availability of a finished, high-quality genome assembly. The finished clone-based assembly of the mouse strain C57BL/6J reported here has over 175,000 fewer gaps and over 139 Mb more of novel sequence, compared with the earlier MGSCv3 draft genome assembly. In a comprehensive analysis of this revised genome sequence, we are now able to define 20,210 protein-coding genes, over a thousand more than predicted in the human genome (19,042 genes). In addition, we identified 439 long, non-proteincoding RNAs with evidence for transcribed orthologs in human. We analyzed the complex and repetitive landscape of 267 Mb of sequence that was missing or misassembled in the previously published assembly, and we provide insights into the reasons for its resistance to sequencing and assembly by whole-genome shotgun approaches. Duplicated regions within newly assembled sequence tend to be of more recent ancestry than duplicates in the published draft, correcting our initial understanding of recent evolution on the mouse lineage. These duplicates appear to be largely composed of sequence regions containing transposable elements and duplicated protein-coding genes; of these, some may be fixed in the mouse population, but at least 40% of segmentally duplicated sequences are copy number variable even among laboratory mouse strains. Mouse lineage-specific regions contain 3,767 genes drawn mainly from rapidly-changing gene families associated with reproductive functions. The finished mouse genome assembly, therefore, greatly improves our understanding of rodent-specific biology and allows the delineation of ancestral biological functions that are shared with human from derived functions that are not.

ABSTRACT Rhodospirillum rubrum is a phototrophic purple nonsulfur bacterium known for its unique and well-studied nitrogen fixation and carbon monoxide oxidation systems and as a source of hydrogen and biodegradable plastic production. To better understand this organism and to facilitate assembly of its sequence, three whole-genome restriction endonuclease maps (XbaI, NheI, and HindIII) of R. rubrum strain ATCC 11170 were created by optical mapping. Optical mapping is a system for creating whole-genome ordered restriction endonuclease maps from randomly sheared genomic DNA molecules extracted from cells. During the sequence finishing process, all three optical maps confirmed a putative error in sequence assembly, while the HindIII map acted as a scaffold for high-resolution alignment with sequence contigs spanning the whole genome. In addition to highlighting optical mapping's role in the assembly and confirmation of genome sequence, this work underscores the unique niche in resolution occupied by the optical mapping system. With a resolution ranging from 6.5 kb (previously published) to 45 kb (reported here), optical mapping advances a “molecular cytogenetics” approach to solving problems in genomic analysis.

Leishmania is a group of protozoan parasites which causes a broad spectrum of diseases resulting in widespread human suffering and death, as well as economic loss from the infection of some domestic animals and wildlife. To further understand the fundamental genomic architecture of this parasite, and to accelerate the on-going sequencing project, a whole-genome XbaI restriction map was constructed using the optical mapping system. This map supplemented traditional physical maps that were generated by fingerprinting and hybridization of cosmid and P1 clone libraries. Thirty-six optical map contigs were constructed for the corresponding known 36 chromosomes of the Leishmania major Friedlin genome. The chromosome sizes ranged from 326.9 to 2821.3 kb, with a total genome size of 34.7 Mb; the average XbaI restriction fragment was 25.3 kb, and ranged from 15.7 to 77.8 kb on a per chromosomes basis. Comparison between the optical maps and the in silico maps of sequence drawn from completed, nearly finished, or large sequence contigs showed that optical maps served several useful functions within the path to create finished sequence by: guiding aspects of the sequence assembly, identifying misassemblies, detection of cosmid or PAC clones misplacements to chromosomes, and validation of sequence stemming from varying degrees of finishing. Our results also showed the potential use of optical maps as a means to detect and characterize map segmental duplication within genomes.

Single molecule approaches offer the promise of large, exquisitely miniature ensembles for the generation of equally large data sets. Although microfluidic devices have previously been designed to manipulate single DNA molecules, many of the functionalities they embody are not applicable to very large DNA molecules, normally extracted from cells. Importantly, such microfluidic devices must work within an integrated system to enable high-throughput biological or biochemical analysis-a key measure of any device aimed at the chemical/biological interface and required if large data sets are to be created for subsequent analysis. The challenge here was to design an integrated microfluidic device to control the deposition or elongation of large DNA molecules (up to millimeters in length), which would serve as a general platform for biological/biochemical analysis to function within an integrated system that included massively parallel data collection and analysis. The approach we took was to use replica molding to construct silastic devices to consistently deposit oriented, elongated DNA molecules onto charged surfaces, creating massive single molecule arrays, which we analyzed for both physical and biochemical insights within an integrated environment that created large data sets. The overall efficacy of this approach was demonstrated by the restriction enzyme mapping and identification of single human genomic DNA molecules.

ABSTRACT Modern comparative genomics has been established, in part, by the sequencing and annotation of a broad range of microbial species. To gain further insights, new sequencing efforts are now dealing with the variety of strains or isolates that gives a species definition and range; however, this number vastly outstrips our ability to sequence them. Given the availability of a large number of microbial species, new whole genome approaches must be developed to fully leverage this information at the level of strain diversity that maximize discovery. Here, we describe how optical mapping, a single-molecule system, was used to identify and annotate chromosomal alterations between bacterial strains represented by several species. Since whole-genome optical maps are ordered restriction maps, sequenced strains of Shigella flexneri serotype 2a (2457T and 301), Yersinia pestis (CO 92 and KIM), and Escherichia coli were aligned as maps to identify regions of homology and to further characterize them as possible insertions, deletions, inversions, or translocations. Importantly, an unsequenced Shigella flexneri strain (serotype Y strain AMC[328Y]) was optically mapped and aligned with two sequenced ones to reveal one novel locus implicated in serotype conversion and several other loci containing insertion sequence elements or phage-related gene insertions. Our results suggest that genomic rearrangements and chromosomal breakpoints are readily identified and annotated against a prototypic sequenced strain by using the tools of optical mapping.

Rhodobacter sphaeroides 2.4.1 is a facultative photoheterotrophic bacterium with tremendous metabolic diversity, which has significantly contributed to our understanding of the molecular genetics of photosynthesis, photoheterotrophy, nitrogen fixation, hydrogen metabolism, carbon dioxide fixation, taxis, and tetrapyrrole biosynthesis. To further understand this remarkable bacterium, and to accelerate an ongoing sequencing project, two whole-genome restriction maps (EcoRI and HindIII) of R. sphaeroides strain 2.4.1 were constructed using shotgun optical mapping. The approach directly mapped genomic DNA by the random mapping of single molecules. The two maps were used to facilitate sequence assembly by providing an optical scaffold for high-resolution alignment and verification of sequence contigs. Our results show that such maps facilitated the closure of sequence gaps by the early detection of nascent sequence contigs during the course of the whole-genome shotgun sequencing process.

ABSTRACT Yersinia pestis is the causative agent of the bubonic, septicemic, and pneumonic plagues (also known as black death) and has been responsible for recurrent devastating pandemics throughout history. To further understand this virulent bacterium and to accelerate an ongoing sequencing project, two whole-genome restriction maps ( Xho I and Pvu II) of Y. pestis strain KIM were constructed using shotgun optical mapping. This approach constructs ordered restriction maps from randomly sheared individual DNA molecules directly extracted from cells. The two maps served different purposes; the Xho I map facilitated sequence assembly by providing a scaffold for high-resolution alignment, while the Pvu II map verified genome sequence assembly. Our results show that such maps facilitated the closure of sequence gaps and, most importantly, provided a purely independent means for sequence validation. Given the recent advancements to the optical mapping system, increased resolution and throughput are enabling such maps to guide sequence assembly at a very early stage of a microbial sequencing project.