'Gel-Stacks' gently confine or reversibly immobilize arrays of single DNA molecules for manipulation and study.

Large DNA molecules (>20 kb) are difficult analytes prone to breakage during serial manipulations and cannot be 'rescued' as full-length amplicons. Accordingly, to present, modify and analyze arrays of large, single DNA molecules, we created an easily realizable approach offering gentle confinement conditions or immobilization via spermidine condensation for controlled delivery of reagents that support live imaging by epifluorescence microscopy termed 'Gel-Stacks.' Molecules are locally confined between two hydrogel surfaces without covalent tethering to support time-lapse imaging and multistep workflows that accommodate large DNA molecules. With a thin polyacrylamide gel layer covalently bound to a glass surface as the base and swappable, reagent-infused, agarose slabs on top, DNA molecules are stably presented for imaging during reagent delivery by passive diffusion.

A database of restriction maps to expand the utility of bacterial artificial chromosomes.

While Bacterial Artificial Chromosomes libraries were once a key resource for the genomic community, they have been obviated, for sequencing purposes, by long-read technologies. Such libraries may now serve as a valuable resource for manipulating and assembling large genomic constructs. To enhance accessibility and comparison, we have developed a BAC restriction map database. Using information from the National Center for Biotechnology Information's cloneDB FTP site, we constructed a database containing the restriction maps for both uniquely placed and insert-sequenced BACs from 11 libraries covering the recognition sequences of the available restriction enzymes. Along with the database, we generated a set of Python functions to reconstruct the database and more easily access the information within. This data is valuable for researchers simply using BACs, as well as those working with larger sections of the genome in terms of synthetic genes, large-scale editing, and mapping.

Trench field-effect transistors integrated in a microfluidic channel and design considerations for charge detection.

Field-effect transistors (FETs) combined with a microfluidic system allow for the electrical detection of charged materials moving in a microfluidic channel. Here, we demonstrate trench-shaped silicon FETs with the combination of a microfluidic channel that can be used for simultaneous electrical and optical detection of charged fluorescent beads. The n-channel silicon trench FETs have a maximum transconductance of 1.83 × 10-5 S at near-zero gate bias voltage, which is beneficial for the high sensitivity of electrical detection. The optical transparency and physical robustness of the integrated microfluidic channel are achieved by a polydimethylsiloxane (PDMS)/glass hybrid cover combining the good sealing characteristics of PDMS, and the thin and flat properties of glass. Device evaluation methodologies and measurement approaches are also presented demonstrating a synchronized time-lapse imaging and electronic detection of bead transport. The proposed device and design consideration could advance the promise of electronic sensing to measure potential differences induced by charged analytes.

The genome of opportunistic fungal pathogen Fusarium oxysporum carries a unique set of lineage-specific chromosomes.

Fusarium oxysporum is a cross-kingdom fungal pathogen that infects plants and humans. Horizontally transferred lineage-specific (LS) chromosomes were reported to determine host-specific pathogenicity among phytopathogenic F. oxysporum. However, the existence and functional importance of LS chromosomes among human pathogenic isolates are unknown. Here we report four unique LS chromosomes in a human pathogenic strain NRRL 32931, isolated from a leukemia patient. These LS chromosomes were devoid of housekeeping genes, but were significantly enriched in genes encoding metal ion transporters and cation transporters. Homologs of NRRL 32931 LS genes, including a homolog of ceruloplasmin and the genes that contribute to the expansion of the alkaline pH-responsive transcription factor PacC/Rim1p, were also present in the genome of NRRL 47514, a strain associated with Fusarium keratitis outbreak. This study provides the first evidence, to our knowledge, for genomic compartmentalization in two human pathogenic fungal genomes and suggests an important role of LS chromosomes in niche adaptation.

Biophysics and the Genomic Sciences.

It is now rare to find biological, or genetic investigations that do not rely on the tools, data, and thinking drawn from the genomic sciences. Much of this revolution is powered by contemporary sequencing approaches that readily deliver large, genome-wide data sets that not only provide genetic insights but also uniquely report molecular outcomes from experiments that biophysicists are increasingly using for potentiating structural and mechanistic investigations. In this perspective, I describe a path of how biophysical thinking greatly contributed to this revolution in ways that parallel advancements in computer science through discussion of several key inventions, described as "foundational devices." These discussions also point at the future of how biophysics and the genomic sciences may become more finely integrated for empowering new measurement paradigms for biological investigations.

A simple dialysis device for large DNA molecules.

The potential of genomic DNA is realized when new modalities are invented that manipulate large DNAs with minimal breakage or loss of sample. Here, we describe a polydimethylsiloxane-polycarbonate membrane device to remove small molecules from a sample while retaining large DNAs. Dialysis rates dramatically change as DNA size in kb (M) increases and DNA dimensions become comparable to pore size, and chain characteristics go from rod-like to Gaussian. Consequently, we describe empirical rates of dialysis, R, as a function of M as falling into two regimes: DNAs ≤ 1 kb show R(M) ∼e -t/τM (t = time, τM = time constant), while DNAs ≥1.65 kb slowly passage with R(M) ∼M -1.68; such partitioning potentiates single-molecule imaging.

Microscale Objects via Restructuring of Large, Double-Stranded DNA Molecules.

As the interest in DNA nanotechnology increases, so does the need for larger and more complex DNA structures. In this work, we describe two methods of using large, double-stranded (ds) DNA to self-assemble sequence-specific, nonrepetitive microscale structures. A model system restructures T7 DNA (40 kb) through sequence-specific biotinylation followed by intramolecular binding to a 40 nm diameter neutravidin bead to create T7 "rosettes". This model system informed the creation of "nodal DNA" where "nodes" with single-stranded DNA flaps are attached to a large dsDNA insert so that a complementary oligonucleotide "strap" bridges the two nodes for restructuring to form a DNA "bolo". To do this in high yield, several methodologies were developed, including a protection/deprotection scheme using RNA/RNase H and dialysis chambers, which remove excess straps while retaining large DNA molecules. To assess these restructuring processes, the DNA was adsorbed onto supported lipid bilayers, allowing for a visual assay of their structure using single-molecule fluorescence microscopy. Good agreement between the expected and observed fluorescence intensity measurements of the individual features of restructured DNA for both the DNA rosettes and bolos gives us a high degree of confidence that both processes give sequence-specific restructuring of large, dsDNA molecules to create microscale objects.

Discrete and Continuum Models for the Salt in Crowded Environments of Suspended Charged Particles.

Electrostatic forces greatly affect the overall dynamics and diffusional activities of suspended charged particles in crowded environments. Accordingly, the concentration of counter- or co-ions in a fluid-''the salt"-determines the range, strength, and order of electrostatic interactions between particles. This environment fosters engineering routes for controlling directed assembly of particles at both the micro- and nanoscale. Here, we analyzed two computational modeling schemes that considered salt within suspensions of charged particles, or polyelectrolytes: discrete and continuum. Electrostatic interactions were included through a Green's function formalism, where the confined fundamental solution for Poisson's equation is resolved by the general geometry Ewald-like method. For the discrete model, the salt was considered as regularized point-charges with a specific valence and size, while concentration fields were defined for each ionic species for the continuum model. These considerations were evolved using Brownian dynamics of the suspended charged particles and the discrete salt ions, while a convection-diffusion transport equation, including the Nernst-Planck diffusion mechanism, accounted for the dynamics of the concentration fields. The salt/particle models were considered as suspensions under slit-confinement conditions for creating crowded "macro-ions", where density distributions and radial distribution functions were used to compare and differentiate computational models. Importantly, our analysis shows that disparate length scales or increased system size presented by the salt and suspended particles are best dealt with using concentration fields to model the ions. These findings were then validated by novel simulations of a semipermeable polyelectrolyte membrane, at the mesoscale, from which ionic channels emerged and enable ion conduction.

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.

In silico evidence for sequence-dependent nucleosome sliding.

Nucleosomes represent the basic building block of chromatin and provide an important mechanism by which cellular processes are controlled. The locations of nucleosomes across the genome are not random but instead depend on both the underlying DNA sequence and the dynamic action of other proteins within the nucleus. These processes are central to cellular function, and the molecular details of the interplay between DNA sequence and nucleosome dynamics remain poorly understood. In this work, we investigate this interplay in detail by relying on a molecular model, which permits development of a comprehensive picture of the underlying free energy surfaces and the corresponding dynamics of nucleosome repositioning. The mechanism of nucleosome repositioning is shown to be strongly linked to DNA sequence and directly related to the binding energy of a given DNA sequence to the histone core. It is also demonstrated that chromatin remodelers can override DNA-sequence preferences by exerting torque, and the histone H4 tail is then identified as a key component by which DNA-sequence, histone modifications, and chromatin remodelers could in fact be coupled.

Gapless genome assembly of Colletotrichum higginsianum reveals chromosome structure and association of transposable elements with secondary metabolite gene clusters.

BACKGROUND: The ascomycete fungus Colletotrichum higginsianum causes anthracnose disease of brassica crops and the model plant Arabidopsis thaliana. Previous versions of the genome sequence were highly fragmented, causing errors in the prediction of protein-coding genes and preventing the analysis of repetitive sequences and genome architecture. RESULTS: Here, we re-sequenced the genome using single-molecule real-time (SMRT) sequencing technology and, in combination with optical map data, this provided a gapless assembly of all twelve chromosomes except for the ribosomal DNA repeat cluster on chromosome 7. The more accurate gene annotation made possible by this new assembly revealed a large repertoire of secondary metabolism (SM) key genes (89) and putative biosynthetic pathways (77 SM gene clusters). The two mini-chromosomes differed from the ten core chromosomes in being repeat- and AT-rich and gene-poor but were significantly enriched with genes encoding putative secreted effector proteins. Transposable elements (TEs) were found to occupy 7% of the genome by length. Certain TE families showed a statistically significant association with effector genes and SM cluster genes and were transcriptionally active at particular stages of fungal development. All 24 subtelomeres were found to contain one of three highly-conserved repeat elements which, by providing sites for homologous recombination, were probably instrumental in four segmental duplications. CONCLUSION: The gapless genome of C. higginsianum provides access to repeat-rich regions that were previously poorly assembled, notably the mini-chromosomes and subtelomeres, and allowed prediction of the complete SM gene repertoire. It also provides insights into the potential role of TEs in gene and genome evolution and host adaptation in this asexual pathogen.

A gapless genome sequence of the fungus Botrytis cinerea.

Following earlier incomplete and fragmented versions of a genome sequence for the grey mould Botrytis cinerea, a gapless, near-finished genome sequence for B. cinerea strain B05.10 is reported. The assembly comprised 18 chromosomes and was confirmed by an optical map and a genetic map based on approximately 75 000 single nucleotide polymorphism (SNP) markers. All chromosomes contained fully assembled centromeric regions, and 10 chromosomes had telomeres on both ends. The genetic map consisted of 4153 cM and a comparison of the genetic distances with the physical distances identified 40 recombination hotspots. The linkage map also identified two mutations, located in the previously described genes Bos1 and BcsdhB, that conferred resistance to the fungicides boscalid and iprodione. The genome was predicted to encode 11 701 proteins. RNAseq data from >20 different samples were used to validate and improve gene models. Manual curation of chromosome 1 revealed interesting features, such as the occurrence of a dicistronic transcript and fully overlapping genes in opposite orientations, as well as many spliced antisense transcripts. Manual curation also revealed that the untranslated regions (UTRs) of genes can be complex and long, with many UTRs exceeding lengths of 1 kb and possessing multiple introns. Community annotation is in progress.

Tension-Dependent Free Energies of Nucleosome Unwrapping.

Nucleosomes form the basic unit of compaction within eukaryotic genomes, and their locations represent an important, yet poorly understood, mechanism of genetic regulation. Quantifying the strength of interactions within the nucleosome is a central problem in biophysics and is critical to understanding how nucleosome positions influence gene expression. By comparing to single-molecule experiments, we demonstrate that a coarse-grained molecular model of the nucleosome can reproduce key aspects of nucleosome unwrapping. Using detailed simulations of DNA and histone proteins, we calculate the tension-dependent free energy surface corresponding to the unwrapping process. The model reproduces quantitatively the forces required to unwrap the nucleosome and reveals the role played by electrostatic interactions during this process. We then demonstrate that histone modifications and DNA sequence can have significant effects on the energies of nucleosome formation. Most notably, we show that histone tails contribute asymmetrically to the stability of the outer and inner turn of nucleosomal DNA and that depending on which histone tails are modified, the tension-dependent response is modulated differently.

Genome Sequence and Annotation of Colletotrichum higginsianum, a Causal Agent of Crucifer Anthracnose Disease.

Colletotrichum higginsianum is an ascomycete fungus causing anthracnose disease on numerous cultivated plants in the family Brassicaceae, as well as the model plant Arabidopsis thaliana We report an assembly of the nuclear genome and gene annotation of this pathogen, which was obtained using a combination of PacBio long-read sequencing and optical mapping.

Allele-Specific Quantification of Structural Variations in Cancer Genomes.

Aneuploidy and structural variations (SVs) generate cancer genomes containing a mixture of rearranged genomic segments with extensive somatic copy number alterations. However, existing methods can identify either SVs or allele-specific copy number alterations but not both simultaneously, which provides a limited view of cancer genome structure. Here, we introduce Weaver, an algorithm for the quantification and analysis of allele-specific copy numbers of SVs. Weaver uses a Markov random field to estimate joint probabilities of allele-specific copy numbers of SVs and their inter-connectivity based on paired-end whole-genome sequencing data. Weaver also predicts the timing of SVs relative to chromosome amplifications. We demonstrate the accuracy of Weaver using simulations and findings from whole-genome optical mapping. We apply Weaver to generate allele-specific copy numbers of SVs for MCF-7 and HeLa cell lines and identify recurrent SV patterns in 44 TCGA ovarian cancer whole-genome sequencing datasets. Our approach provides a more complete assessment of the complex genomic architectures inherent to many cancer genomes.

Maligner: a fast ordered restriction map aligner.

MOTIVATION: The Optical Mapping System discovers structural variants and potentiates sequence assembly of genomes via scaffolding and comparisons that globally validate or correct sequence assemblies. Despite its utility, there are few publicly available tools for aligning optical mapping datasets. RESULTS: Here we present software, named 'Maligner', for the alignment of both single molecule restriction maps (Rmaps) and in silico restriction maps of sequence contigs to a reference. Maligner provides two modes of alignment: an efficient, sensitive dynamic programming implementation that scales to large eukaryotic genomes, and a faster indexed based implementation for finding alignments with unmatched sites in the reference but not the query. We compare our software to other publicly available tools on Rmap datasets and show that Maligner finds more correct alignments in comparable runtime. Lastly, we introduce the M-Score statistic for normalizing alignment scores across restriction maps and demonstrate its utility for selecting high quality alignments. AVAILABILITY AND IMPLEMENTATION: The Maligner software is written in C ++ and is available at https://github.com/LeeMendelowitz/maligner under the GNU General Public License. CONTACT: mpop@umiacs.umd.edu.

DNA binding fluorescent proteins for the direct visualization of large DNA molecules.

Fluorescent proteins that also bind DNA molecules are useful reagents for a broad range of biological applications because they can be optically localized and tracked within cells, or provide versatile labels for in vitro experiments. We report a novel design for a fluorescent, DNA-binding protein (FP-DBP) that completely 'paints' entire DNA molecules, whereby sequence-independent DNA binding is accomplished by linking a fluorescent protein to two small peptides (KWKWKKA) using lysine for binding to the DNA phosphates, and tryptophan for intercalating between DNA bases. Importantly, this ubiquitous binding motif enables fluorescent proteins (Kd = 14.7 µM) to confluently stain DNA molecules and such binding is reversible via pH shifts. These proteins offer useful robust advantages for single DNA molecule studies: lack of fluorophore mediated photocleavage and staining that does not perturb polymer contour lengths. Accordingly, we demonstrate confluent staining of naked DNA molecules presented within microfluidic devices, or localized within live bacterial cells.

Comparative Genomic Analyses of the Human NPHP1 Locus Reveal Complex Genomic Architecture and Its Regional Evolution in Primates.

Many loci in the human genome harbor complex genomic structures that can result in susceptibility to genomic rearrangements leading to various genomic disorders. Nephronophthisis 1 (NPHP1, MIM# 256100) is an autosomal recessive disorder that can be caused by defects of NPHP1; the gene maps within the human 2q13 region where low copy repeats (LCRs) are abundant. Loss of function of NPHP1 is responsible for approximately 85% of the NPHP1 cases-about 80% of such individuals carry a large recurrent homozygous NPHP1 deletion that occurs via nonallelic homologous recombination (NAHR) between two flanking directly oriented ~45 kb LCRs. Published data revealed a non-pathogenic inversion polymorphism involving the NPHP1 gene flanked by two inverted ~358 kb LCRs. Using optical mapping and array-comparative genomic hybridization, we identified three potential novel structural variant (SV) haplotypes at the NPHP1 locus that may protect a haploid genome from the NPHP1 deletion. Inter-species comparative genomic analyses among primate genomes revealed massive genomic changes during evolution. The aggregated data suggest that dynamic genomic rearrangements occurred historically within the NPHP1 locus and generated SV haplotypes observed in the human population today, which may confer differential susceptibility to genomic instability and the NPHP1 deletion within a personal genome. Our study documents diverse SV haplotypes at a complex LCR-laden human genomic region. Comparative analyses provide a model for how this complex region arose during primate evolution, and studies among humans suggest that intra-species polymorphism may potentially modulate an individual's susceptibility to acquiring disease-associated alleles.

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.