Variation in genome structure is an important source of human genetic polymorphism: It affects a large proportion of the genome and has a variety of phenotypic consequences relevant to health and disease. In spite of this, human genome structure variation is incompletely characterized due to a lack of approaches for discovering a broad range of structural variants in a global, comprehensive fashion. We addressed this gap with Optical Mapping, a high-throughput, high-resolution single-molecule system for studying genome structure. We used Optical Mapping to create genome-wide restriction maps of a complete hydatidiform mole and three lymphoblast-derived cell lines, and we validated the approach by demonstrating a strong concordance with existing methods. We also describe thousands of new variants with sizes ranging from kb to Mb.

The recent plethora of sequenced genomes has just ushered in a new era of genetics-based research. Although an impressive number of species have been, or are planned to be sequenced, the full value of such efforts will be fully accrued when patterns of variation can be discerned and annotated for many strains or isolates within a given species. As such, bacteria are an ideal place to start investigations aimed at the discernment of genome rearrangements, chromosome deletions, and horizontal transfer of foreign DNA, since these events help drive bacterial evolution. For example, genome remodeling events may cause irreversible gene loss, or add novel functionalities to an organism. Unfortunately, current approaches do not adequately identify and characterize such large-scale genomic rearrangements. The Optical Mapping System, developed in our laboratory creates high resolution maps of entire genomes, using DNA directly extracted from cells—this approach obviates the need for libraries, PCR, and probe technologies. The system uses a complex blend of single molecule technologies to enable high throughput and the construction of reliable maps. This capability has been proven by the construction and sequence comparison of 13 bacterial optical maps, and 3 parasites. Recent advances in both throughput and resolution of the Optical Mapping System has enabled genomic comparisons amongst different strains of the same species or closely related species, allowing for the pinpoint discernment of insertions, deletions and rearrangements. Comparisons of optical maps vs. in silico maps, and in silico vs. in silico maps constructed for two strains of Yersinia pestis (CO-92 biovar Orientalis and KIM), E. coli K12 and Shigella flexneri 2a, two strains of S. flexneri (2a and Y), and two strains of Rhodobacter sphaeroides (2.4.1 and ATCC 17029) have revealed regions of homology, insertion sites and a panoply of rearrangements. These results portend the wide use of Optical Mapping to uniquely provide genome structural details for a large number of strains, isolates or even closely related species, in ways that would complement direct sequence analysis.

About 85% of the maize genome consists of highly repetitive sequences that are interspersed by low-copy, gene-coding sequences. The maize community has dealt with this genomic complexity by the construction of an integrated genetic and physical map (iMap), but this resource alone was not sufficient for ensuring the quality of the current sequence build. For this purpose, we constructed a genome-wide, high-resolution optical map of the maize inbred line B73 genome containing >91,000 restriction sites (averaging 1 site/ approximately 23 kb) accrued from mapping genomic DNA molecules. Our optical map comprises 66 contigs, averaging 31.88 Mb in size and spanning 91.5% (2,103.93 Mb/ approximately 2,300 Mb) of the maize genome. A new algorithm was created that considered both optical map and unfinished BAC sequence data for placing 60/66 (2,032.42 Mb) optical map contigs onto the maize iMap. The alignment of optical maps against numerous data sources yielded comprehensive results that proved revealing and productive. For example, gaps were uncovered and characterized within the iMap, the FPC (fingerprinted contigs) map, and the chromosome-wide pseudomolecules. Such alignments also suggested amended placements of FPC contigs on the maize genetic map and proactively guided the assembly of chromosome-wide pseudomolecules, especially within complex genomic regions. Lastly, we think that the full integration of B73 optical maps with the maize iMap would greatly facilitate maize sequence finishing efforts that would make it a valuable reference for comparative studies among cereals, or other maize inbred lines and cultivars.

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.

Background: Methylation of CpG dinucleotides is a fundamental mechanism of epigenetic regulation in eukaryotic genomes. Development of methods for rapid genome wide methylation profiling will greatly facilitate both hypothesis and discovery driven research in the field of epigenetics. In this regard, a single molecule approach to methylation profiling offers several unique advantages that include elimination of chemical DNA modification steps and PCR amplification.Results: A single molecule approach is presented for the discernment of methylation profiles, based on optical mapping. We report results from a series of pilot studies demonstrating the capabilities of optical mapping as a platform for methylation profiling of whole genomes. Optical mapping was used to discern the methylation profile from both an engineered and wild type Escherichia coli. Furthermore, the methylation status of selected loci within the genome of human embryonic stem cells was profiled using optical mapping.Conclusion: The optical mapping platform effectively detects DNA methylation patterns. Due to single molecule detection, optical mapping offers significant advantages over other technologies. This advantage stems from obviation of DNA modification steps, such as bisulfite treatment, and the ability of the platform to assay repeat dense regions within mammalian genomes inaccessible to techniques using array-hybridization technologies.

Abstract Background Rice feeds much of the world, and possesses the simplest genome analyzed to date within the grass family, making it an economically relevant model system for other cereal crops. Although the rice genome is sequenced, validation and gap closing efforts require purely independent means for accurate finishing of sequence build data. Results To facilitate ongoing sequencing finishing and validation efforts, we have constructed a whole-genome SwaI optical restriction map of the rice genome. The physical map consists of 14 contigs, covering 12 chromosomes, with a total genome size of 382.17 Mb; this value is about 11% smaller than original estimates. 9 of the 14 optical map contigs are without gaps, covering chromosomes 1, 2, 3, 4, 5, 7, 8 10, and 12 in their entirety – including centromeres and telomeres. Alignments between optical and in silico restriction maps constructed from IRGSP (International Rice Genome Sequencing Project) and TIGR (The Institute for Genomic Research) genome sequence sources are comprehensive and informative, evidenced by map coverage across virtually all published gaps, discovery of new ones, and characterization of sequence misassemblies; all totalling ~14 Mb. Furthermore, since optical maps are ordered restriction maps, identified discordances are pinpointed on a reliable physical scaffold providing an independent resource for closure of gaps and rectification of misassemblies. Conclusion Analysis of sequence and optical mapping data effectively validates genome sequence assemblies constructed from large, repeat-rich genomes. Given this conclusion we envision new applications of such single molecule analysis that will merge advantages offered by high-resolution optical maps with inexpensive, but short sequence reads generated by emerging sequencing platforms. Lastly, map construction techniques presented here points the way to new types of comparative genome analysis that would focus on discernment of structural differences revealed by optical maps constructed from a broad range of rice subspecies and varieties.