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.

Rhodopseudomonas palustris is a photosynthetic bacterium that can use many forms of carbon, nitrogen, and electron donors. Under anaerobic conditions it can generate energy from light and convert nitrogen gas to ammonia and hydrogen (a biofuel) by nitrogen fixation. A striking feature of the genome sequence of R. palustris CGA009 is genes encoding three different nitrogenases and the accessory proteins needed for nitrogenase assembly. AnfHDGK, nifHDK, and vnfHDGK genes encode iron (Fe)-containing, molybdenum (Mo)-containing, and vanadium (V)-containing nitrogenases. To address the question of how R. palustris differentially regulates nitrogenase gene expression, we constructed anfHnifH, anfHvnfH, and nifHvnfH double mutants and analyzed the whole genome gene expression profiles of each mutant and wild-type cells grown under nitrogen-fixing conditions. Each mutant expressed a single functional nitrogenase (Mo, V or Fe) in a minimal medium that contained molybdenum and other trace elements. Wild-type and the Mo-nitrogenase active mutant cells expressed over 150 genes at levels of 2-fold or higher when grown under nitrogen-fixing conditions as compared to when grown with ammonia. Among these were the 30 genes in the nif gene cluster, which were expressed at 5to 200-fold higher (depending on the gene) levels in cells grown under nitrogen-fixing conditions. Genes in the anf and vnf clusters were not expressed. By contrast, cells with an active Fe-nitrogenase only or an active V-nitrogenase only expressed all of the nif genes (except nifH which was deleted), all of the anf genes, and all of the vnf genes. It makes sense that nif genes would be expressed because many of them are needed for the assembly of the Feand V-nitrogenases. These results indicate that R. palustris synthesizes both its Feand V-nitrogenases in situations where it is unable to synthesize an active Mo-nitrogenase. The mechanism by which Feand V-nitrogenase gene expression is activated is not known, but does not involve relief of Mo repression.

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.