Electrostatic confinement and manipulation of DNA molecules for genome analysis.

Very large DNA molecules enable comprehensive analysis of complex genomes, such as human, cancer, and plants because they span across sequence repeats and complex somatic events. When physically manipulated, or analyzed as single molecules, long polyelectrolytes are problematic because of mechanical considerations that include shear-mediated breakage, dealing with the massive size of these coils, or the length of stretched DNAs using common experimental techniques and fluidic devices. Accordingly, we harness analyte "issues" as exploitable advantages by our invention and characterization of the "molecular gate," which controls and synchronizes formation of stretched DNA molecules as DNA dumbbells within nanoslit geometries. Molecular gate geometries comprise micro- and nanoscale features designed to synergize very low ionic strength conditions in ways we show effectively create an "electrostatic bottle." This effect greatly enhances molecular confinement within large slit geometries and supports facile, synchronized electrokinetic loading of nanoslits, even without dumbbell formation. Device geometries were considered at the molecular and continuum scales through computer simulations, which also guided our efforts to optimize design and functionalities. In addition, we show that the molecular gate may govern DNA separations because DNA molecules can be electrokinetically triggered, by varying applied voltage, to enter slits in a size-dependent manner. Lastly, mapping the Mesoplasmaflorum genome, via synchronized dumbbell formation, validates our nascent approach as a viable starting point for advanced development that will build an integrated system capable of large-scale genome analysis.

A clone-free, single molecule map of the domestic cow (Bos taurus) genome.

BACKGROUND: The cattle (Bos taurus) genome was originally selected for sequencing due to its economic importance and unique biology as a model organism for understanding other ruminants, or mammals. Currently, there are two cattle genome sequence assemblies (UMD3.1 and Btau4.6) from groups using dissimilar assembly algorithms, which were complemented by genetic and physical map resources. However, past comparisons between these assemblies revealed substantial differences. Consequently, such discordances have engendered ambiguities when using reference sequence data, impacting genomic studies in cattle and motivating construction of a new optical map resource--BtOM1.0--to guide comparisons and improvements to the current sequence builds. Accordingly, our comprehensive comparisons of BtOM1.0 against the UMD3.1 and Btau4.6 sequence builds tabulate large-to-immediate scale discordances requiring mediation. RESULTS: The optical map, BtOM1.0, spanning the B. taurus genome (Hereford breed, L1 Dominette 01449) was assembled from an optical map dataset consisting of 2,973,315 (439 X; raw dataset size before assembly) single molecule optical maps (Rmaps; 1 Rmap = 1 restriction mapped DNA molecule) generated by the Optical Mapping System. The BamHI map spans 2,575.30 Mb and comprises 78 optical contigs assembled by a combination of iterative (using the reference sequence: UMD3.1) and de novo assembly techniques. BtOM1.0 is a high-resolution physical map featuring an average restriction fragment size of 8.91 Kb. Comparisons of BtOM1.0 vs. UMD3.1, or Btau4.6, revealed that Btau4.6 presented far more discordances (7,463) vs. UMD3.1 (4,754). Overall, we found that Btau4.6 presented almost double the number of discordances than UMD3.1 across most of the 6 categories of sequence vs. map discrepancies, which are: COMPLEX (misassembly), DELs (extraneous sequences), INSs (missing sequences), ITs (Inverted/Translocated sequences), ECs (extra restriction cuts) and MCs (missing restriction cuts). CONCLUSION: Alignments of UMD3.1 and Btau4.6 to BtOM1.0 reveal discordances commensurate with previous reports, and affirm the NCBI's current designation of UMD3.1 sequence assembly as the "reference assembly" and the Btau4.6 as the "alternate assembly." The cattle genome optical map, BtOM1.0, when used as a comprehensive and largely independent guide, will greatly assist improvements to existing sequence builds, and later serve as an accurate physical scaffold for studies concerning the comparative genomics of cattle breeds.

Single-molecule analysis reveals widespread structural variation in multiple myeloma.

Multiple myeloma (MM), a malignancy of plasma cells, is characterized by widespread genomic heterogeneity and, consequently, differences in disease progression and drug response. Although recent large-scale sequencing studies have greatly improved our understanding of MM genomes, our knowledge about genomic structural variation in MM is attenuated due to the limitations of commonly used sequencing approaches. In this study, we present the application of optical mapping, a single-molecule, whole-genome analysis system, to discover new structural variants in a primary MM genome. Through our analysis, we have identified and characterized widespread structural variation in this tumor genome. Additionally, we describe our efforts toward comprehensive characterization of genome structure and variation by integrating our findings from optical mapping with those from DNA sequencing-based genomic analysis. Finally, by studying this MM genome at two time points during tumor progression, we have demonstrated an increase in mutational burden with tumor progression at all length scales of variation.

Discovery of structural alterations in solid tumor oligodendroglioma by single molecule analysis.

BACKGROUND: Solid tumors present a panoply of genomic alterations, from single base changes to the gain or loss of entire chromosomes. Although aberrations at the two extremes of this spectrum are readily defined, comprehensive discernment of the complex and disperse mutational spectrum of cancer genomes remains a significant challenge for current genome analysis platforms. In this context, high throughput, single molecule platforms like Optical Mapping offer a unique perspective. RESULTS: Using measurements from large ensembles of individual DNA molecules, we have discovered genomic structural alterations in the solid tumor oligodendroglioma. Over a thousand structural variants were identified in each tumor sample, without any prior hypotheses, and often in genomic regions deemed intractable by other technologies. These findings were then validated by comprehensive comparisons to variants reported in external and internal databases, and by selected experimental corroborations. Alterations range in size from under 5 kb to hundreds of kilobases, and comprise insertions, deletions, inversions and compound events. Candidate mutations were scored at sub-genic resolution and unambiguously reveal structural details at aberrant loci. CONCLUSIONS: The Optical Mapping system provides a rich description of the complex genomes of solid tumors, including sequence level aberrations, structural alterations and copy number variants that power generation of functional hypotheses for oligodendroglioma genetics.

High-resolution human genome structure by single-molecule analysis.

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.

Engineering BspQI nicking enzymes and application of N.BspQI in DNA labeling and production of single-strand DNA.

BspQI is a thermostable Type IIS restriction endonuclease (REase) with the recognition sequence 5'GCTCTTC N1/N4 3'. Here we report the cloning and expression of the bspQIR gene for the BspQI restriction enzyme in Escherichia coli. Alanine scanning of the BspQI charged residues identified a number of DNA nicking variants. After sampling combinations of different amino acid substitutions, an Nt.BspQI triple mutant (E172A/E248A/E255K) was constructed with predominantly top-strand DNA nicking activity. Furthermore, a triple mutant of BspQI (Nb.BspQI, N235A/K331A/R428A) was engineered to create a bottom-strand nicking enzyme. In addition, we demonstrated the application of Nt.BspQI in optical mapping of single DNA molecules. Nt or Nb.BspQI-nicked dsDNA can be further digested by E. coli exonuclease III to create ssDNA for downstream applications. BspQI contains two potential catalytic sites: a top-strand catalytic site (Ct) with a D-H-N-K motif found in the HNH endonuclease family and a bottom-strand catalytic site (Cb) with three scattered Glu residues. BlastP analysis of proteins in GenBank indicated a putative restriction enzyme with significant amino acid sequence identity to BspQI from the sequenced bacterial genome Croceibacter atlanticus HTCC2559. This restriction gene was amplified by PCR and cloned into a T7 expression vector. Restriction mapping and run-off DNA sequencing of digested products from the partially purified enzyme indicated that it is an EarI isoschizomer with 6-bp recognition, which we named CatHI (CTCTTC N1/N4).

A single molecule scaffold for the maize genome.

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.

Lineage-specific biology revealed by a finished genome assembly of the mouse.

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-protein-coding 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.

Optical mapping discerns genome wide DNA methylation profiles.

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.

An integrative approach for the optical sequencing of single DNA molecules.

A new approach for optically sequencing ensembles of single DNA molecules using DNA polymerase to mediate the consecutive incorporation of fluorochrome-labeled nucleotides into an array of large single DNA molecules is presented. The approach utilizes cycles of labeled fluorochrome addition, detection to count incorporations, and bleaching to reset the counter. These additions are imaged and analyzed to estimate the number of labeled additions and to correlate them on a per-locus basis along DNA backbones. Initial studies used precisely labeled polymerase chain reaction products to aid the development and validation of simple models of fluorochrome point spread functions within the imaging system. In complementary studies, nucleotides labeled with the fluorochrome R110 were incorporated into surface-elongated lambda DNA, and fluorescent signals corresponding to the addition of R110-dUTP were counted and assigned precise loci along DNA backbones. The labeled DNAs were then subjected to photobleaching and to a second cycle of addition of R110-labeled nucleotides-a second round of additions was correlated with the first to establish strings of addition histories among the ensemble of largely double-stranded templates. These results confirm the basic operational validity of this approach and point the way to the development of a practical system for optical sequencing.