Single-molecule optical genome mapping in nanochannels: multidisciplinarity at the nanoscale

Abstract The human genome contains multiple layers of information that extend beyond the genetic sequence. In fact, identical genetics do not necessarily yield identical phenotypes as evident for the case of two different cell types in the human body. The great variation in structure and function displayed by cells with identical genetic background is attributed to additional genomic information content. This includes large-scale genetic aberrations, as well as diverse epigenetic patterns that are crucial for regulating specific cell functions. These genetic and epigenetic patterns operate in concert in order to maintain specific cellular functions in health and disease. Single-molecule optical genome mapping is a high-throughput genome analysis method that is based on imaging long chromosomal fragments stretched in nanochannel arrays. The access to long DNA molecules coupled with fluorescent tagging of various genomic information presents a unique opportunity to study genetic and epigenetic patterns in the genome at a single-molecule level over large genomic distances. Optical mapping entwines synergistically chemical, physical, and computational advancements, to uncover invaluable biological insights, inaccessible by sequencing technologies. Here we describe the method’s basic principles of operation, and review the various available mechanisms to fluorescently tag genomic information. We present some of the recent biological and clinical impact enabled by optical mapping and present recent approaches for increasing the method’s resolution and accuracy. Finally, we discuss how multiple layers of genomic information may be mapped simultaneously on the same DNA molecule, thus paving the way for characterizing multiple genomic observables on individual DNA molecules.

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Single-molecule analysis reveals widespread structural variation in multiple myeloma

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1418577112 ◽

2015 ◽

Vol 112 (25) ◽

pp. 7689-7694 ◽

Cited By ~ 29

Author(s):

Aditya Gupta ◽

Michael Place ◽

Steven Goldstein ◽

Deepayan Sarkar ◽

Shiguo Zhou ◽

...

Keyword(s):

Multiple Myeloma ◽

Tumor Progression ◽

Single Molecule ◽

Large Scale ◽

Structural Variation ◽

Plasma Cells ◽

Optical Mapping ◽

Genome Structure ◽

Genomic Analysis ◽

Whole Genome Analysis

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.

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Genotype-Phenotype Relations of the von Hippel-Lindau Tumor Suppressor Inferred from a Large-Scale Analysis of Disease Mutations and Interactors

10.1101/405845 ◽

2018 ◽

Author(s):

Giovanni Minervini ◽

Federica Quaglia ◽

Francesco Tabaro ◽

Silvio C.E. Tosatto

Keyword(s):

Tumor Suppressor ◽

Large Scale ◽

Specific Cell ◽

Scale Analysis ◽

Cancer Syndrome ◽

Von Hippel Lindau ◽

Cell Functions ◽

Large Scale Analysis ◽

Tumor Transformation ◽

Cancer Types

AbstractFamiliar cancers represent a privileged point of view for studying the complex cellular events inducing tumor transformation. Von Hippel-Lindau syndrome, a familiar predisposition to develop cancer is a clear example. Here, we present our efforts to decipher the role of von Hippel-Lindau tumor suppressor protein (pVHL) in cancer insurgence. We collected high quality information about both pVHL mutations and interactors to investigate the association between patient phenotypes, mutated protein surface and impaired interactions. Our data suggest that different phenotypes correlate with localized perturbations of the pVHL structure, with specific cell functions associated to different protein surfaces. We propose five different pVHL interfaces to be selectively involved in modulating proteins regulating gene expression, protein homeostasis as well as to address extracellular matrix (ECM) and ciliogenesis associated functions. These data were used to drive molecular docking of pVHL with its interactors and guide Petri net simulations of the most promising alterations. We predict that disruption of pVHL association with certain interactors can trigger tumor transformation, inducing metabolism imbalance and ECM remodeling. Collectively taken, our findings provide novel insights into VHL-associated tumorigenesis. This highly integrated in silico approach may help elucidate novel treatment paradigms for VHL disease.Author summaryCancer is generally caused by a series of mutations accumulating over time in a healthy tissue, which becomes re-programmed to proliferate at the expense of the hosting organism. This process is difficult to follow and understand as events in a multitude of different genes can lead to similar outcomes without apparent cause. The von Hippel-Lindau (VHL) tumor suppressor is one of the few genes harboring a familiar cancer syndrome, i.e. VHL mutations are known to cause a predictable series of events leading cancer in the kidneys and a few selected other tissues. This article describes a large-scale analysis to relate known VHL mutations to specific cancer pathways by looking at the molecular interactions. Different cancer types appear to be caused by mutations changing the surface of specific parts of the VHL protein. By looking at the VHL interactors involved, it is therefore possible to identify other candidate genes for mutations leading to very similar cancer types.

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OptiDiff: structural variation detection from single optical mapping reads

10.1101/2022.01.08.475501 ◽

2022 ◽

Author(s):

Mehmet Akdel ◽

Dick de Ridder

Keyword(s):

Single Molecule ◽

Large Scale ◽

Structural Variation ◽

Optical Mapping ◽

Cost Effective ◽

Sequencing Data ◽

Mapping Technology ◽

Cost Effective Alternative ◽

Algorithmic Approaches ◽

Broad Interest

Detecting structural variation (SV) in eukaryotic genomes is of broad interest due to its often dramatic phenotypic effects, but remains a major, costly challenge based on DNA sequencing data. A cost-effective alternative in detecting large-scale SV has become available with advances in optical mapping technology. However, the algorithmic approaches to identifying SVs from optical mapping data are limited. Here, we propose a novel, open-source SV detection tool, OptiDiff, which employs a single molecule based approach to detect and classify homozygous and heterozygous SVs at coverages as low as 20x, showing better performance than the state of the art.

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Molecular microscopy of DNA for genome analysis: optical mapping of restriction digests

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100136623 ◽

1995 ◽

Vol 53 ◽

pp. 50-51

Author(s):

Edward J. Huff ◽

Weiwen Cai ◽

Xinghua Hu ◽

John Huang ◽

Junping Jing ◽

...

Keyword(s):

Single Molecule ◽

Genome Analysis ◽

Optical Mapping ◽

Ccd Camera ◽

Dna Molecules ◽

Specific Sequence ◽

Single Dna Molecules ◽

Single Clone ◽

Rapid Generation ◽

Glass Surfaces

Optical microscopy of individual DNA molecules has been an interesting technique for the past 15 years, but until recently has not been useful for genome analysis. We have developed Optical Mapping an emerging single molecule approach for the rapid generation of ordered restriction maps. Many identical individual DNA molecules from a single clone are elongated and fixed onto derivatized glass surfaces, digested with a restriction enzyme which cuts the DNA wherever a specific sequence pattern is found, stained with YOYO, and imaged with a cooled CCD camera attached to an automated epi-fluorescence microscope. Images are automatically processed to correct for non-uniform illumination, remove background, locate the DNA fragments, reject objects which do not look like single DNA molecules, recognize which fragments originate from an original uncut molecule, and calculate the relative sizes of the fragments by apparent length and fluorescence intensity. Results from many molecules are combined by clustering to recognize a consistent cutting pattern. Molecules which match the pattern are averaged to improve the sizing accuracy.

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Enrichment of megabase-sized DNA molecules for single-molecule optical mapping and next-generation sequencing

Scientific Reports ◽

10.1038/s41598-017-18091-6 ◽

2017 ◽

Vol 7 (1) ◽

Cited By ~ 2

Author(s):

Joanna M. Łopacińska-Jørgensen ◽

Jonas N. Pedersen ◽

Mads Bak ◽

Mana M. Mehrjouy ◽

Kristian T. Sørensen ◽

...

Keyword(s):

Next Generation Sequencing ◽

Single Molecule ◽

Optical Mapping ◽

Next Generation ◽

Dna Molecules ◽

Generation Sequencing

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Genome mapping resolves structural variation within segmental duplications associated with microdeletion/microduplication syndromes

10.1101/2020.04.30.071449 ◽

2020 ◽

Author(s):

Yulia Mostovoy ◽

Feyza Yilmaz ◽

Stephen K. Chow ◽

Catherine Chu ◽

Chin Lin ◽

...

Keyword(s):

Single Molecule ◽

Structural Variation ◽

Optical Mapping ◽

Genome Mapping ◽

Sequence Similarity ◽

Chromosomal Rearrangements ◽

Population Level ◽

Segmental Duplications ◽

Microdeletion Syndromes ◽

Genomic Disorders

AbstractSegmental duplications (SDs) are a class of long, repetitive DNA elements whose paralogs share a high level of sequence similarity with each other. SDs mediate chromosomal rearrangements that lead to structural variation in the general population as well as genomic disorders associated with multiple congenital anomalies, including the 7q11.23 (Williams-Beuren Syndrome, WBS), 15q13.3, and 16p12.2 microdeletion syndromes. These three genomic regions, and the SDs within them, have been previously analyzed in a small number of individuals. However, population-level studies have been lacking because most techniques used for analyzing these complex regions are both labor- and cost-intensive. In this study, we present a high-throughput technique to genotype complex structural variation using a single molecule, long-range optical mapping approach. We identified novel structural variants (SVs) at 7q11.23, 15q13.3 and 16p12.2 using optical mapping data from 154 phenotypically normal individuals from 26 populations comprising 5 super-populations. We detected several novel SVs for each locus, some of which had significantly different prevalence between populations. Additionally, we refined the microdeletion breakpoints located within complex SDs in two patients with WBS, one patient with 15q13.3, and one patient with 16p12.2 microdeletion syndromes. The population-level data presented here highlights the extreme diversity of large and complex SVs within SD-containing regions. The approach we outline will greatly facilitate the investigation of the role of inter-SD structural variation as a driver of chromosomal rearrangements and genomic disorders.

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Large-scale structural variation detection in subterranean clover subtypes using optical mapping validated at nucleotide level

10.1101/232132 ◽

2017 ◽

Author(s):

Yuxuan Yuan ◽

Zbyněk Milec ◽

Philipp E. Bayer ◽

Jan Vrána ◽

Jaroslav Doležel ◽

...

Keyword(s):

Single Molecule ◽

Large Scale ◽

Optical Mapping ◽

Subterranean Clover ◽

Structural Variations ◽

Recognition Sequence ◽

Nucleotide Level ◽

Total Size ◽

Short Reads ◽

A Genome

AbstractWhole genome sequencing has been widely used to detect structural variations (SVs). However, the limited single molecule size makes it difficult to characterize large-scale SVs in a genome because they cannot fully cover such vast and complex regions. Recently, optical mapping in nanochannels has provided novel resolution to detect large-scale SVs by comparing the physical location of the nickase recognition sequence in genomes. Other than in humans, SVs discovered in plants by optical mapping have not been validated. To assess the accuracy of SV calling in plants by optical mapping, we selected two genetically diverse subspecies of the Trifolium model species, subterranean clover cvs. Daliak and Yarloop. The SVs discovered by BioNano optical mapping (BOM) were validated using Illumina short reads. In the analysis, BOM identified 12 large-scale regions containing deletions and 19 containing insertions in Yarloop. The 12 large-scale regions contained 71 small deletions when validated by Illumina short reads. The results suggest that BOM could detect the total size of deletions and insertions, but it could not precisely report the location and actual quantity of SVs in the genome. Nucleotide-level validation is crucial to confirm and characterize SVs reported by optical mapping. The accuracy of SV detection by BOM is highly dependent on the quality of reference genomes and the density of selected nickases.

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Optical Mapping of Plasmodium falciparum Chromosome 2

Genome Research ◽

10.1101/gr.9.2.175 ◽

1999 ◽

Vol 9 (2) ◽

pp. 175-181 ◽

Cited By ~ 3

Author(s):

Junping Jing ◽

Zhongwu Lai ◽

Christopher Aston ◽

Jieyi Lin ◽

Daniel J. Carucci ◽

...

Keyword(s):

Plasmodium Falciparum ◽

High Resolution ◽

Genome Sequencing ◽

Large Scale ◽

Dna Analysis ◽

Optical Mapping ◽

Chromosome 2 ◽

Dna Molecules ◽

Restriction Maps ◽

Plasmid Library

Detailed restriction maps of microbial genomes are a valuable resource in genome sequencing studies but are toilsome to construct by contig construction of maps derived from cloned DNA. Analysis of genomic DNA enables large stretches of the genome to be mapped and circumvents library construction and associated cloning artifacts. We used pulsed-field gel electrophoresis purified Plasmodium falciparum chromosome 2 DNA as the starting material for optical mapping, a system for making ordered restriction maps from ensembles of individual DNA molecules. DNA molecules were bound to derivatized glass surfaces, cleaved with NheI or BamHI, and imaged by digital fluorescence microscopy. Large pieces of the chromosome containing ordered DNA restriction fragments were mapped. Maps were assembled from 50 molecules producing an average contig depth of 15 molecules and high-resolution restriction maps covering the entire chromosome. Chromosome 2 was found to be 976 kb by optical mapping withNheI, and 946 kb with BamHI, which compares closely to the published size of 947 kb from large-scale sequencing. The maps were used to further verify assemblies from the plasmid library used for sequencing. Maps generated in silico from the sequence data were compared to the optical mapping data, and good correspondence was found. Such high-resolution restriction maps may become an indispensable resource for large-scale genome sequencing projects.

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Metabolic Regulation and Related Molecular Mechanisms in Various Stem Cell Functions

Current Stem Cell Research & Therapy ◽

10.2174/1574888x15666200512105347 ◽

2020 ◽

Vol 15 (6) ◽

pp. 531-546 ◽

Cited By ~ 1

Author(s):

Hwa-Yong Lee ◽

In-Sun Hong

Keyword(s):

Stem Cells ◽

Stem Cell ◽

Oxidative Phosphorylation ◽

Metabolic Pathways ◽

Critical Role ◽

The Self ◽

Specific Cell ◽

Differentiation Potential ◽

Self Renewal ◽

Cell Functions

Recent studies on the mechanisms that link metabolic changes with stem cell fate have deepened our understanding of how specific metabolic pathways can regulate various stem cell functions during the development of an organism. Although it was originally thought to be merely a consequence of the specific cell state, metabolism is currently known to play a critical role in regulating the self-renewal capacity, differentiation potential, and quiescence of stem cells. Many studies in recent years have revealed that metabolic pathways regulate various stem cell behaviors (e.g., selfrenewal, migration, and differentiation) by modulating energy production through glycolysis or oxidative phosphorylation and by regulating the generation of metabolites, which can modulate multiple signaling pathways. Therefore, a more comprehensive understanding of stem cell metabolism could allow us to establish optimal culture conditions and differentiation methods that would increase stem cell expansion and function for cell-based therapies. However, little is known about how metabolic pathways regulate various stem cell functions. In this context, we review the current advances in metabolic research that have revealed functional roles for mitochondrial oxidative phosphorylation, anaerobic glycolysis, and oxidative stress during the self-renewal, differentiation and aging of various adult stem cell types. These approaches could provide novel strategies for the development of metabolic or pharmacological therapies to promote the regenerative potential of stem cells and subsequently promote their therapeutic utility.

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Single bacterial resolvases first exploit, then constrain intrinsic dynamics of the Holliday junction to direct recombination

Nucleic Acids Research ◽

10.1093/nar/gkab096 ◽

2021 ◽

Author(s):

Sujay Ray ◽

Nibedita Pal ◽

Nils G Walter

Keyword(s):

Cluster Analysis ◽

Homologous Recombination ◽

Single Molecule ◽

Holliday Junction ◽

Energetic Cost ◽

Genomic Integrity ◽

Dna Molecules ◽

Single Molecule Fluorescence ◽

And Cluster Analysis ◽

Fluorescence Observation

Abstract Homologous recombination forms and resolves an entangled DNA Holliday Junction (HJ) crucial for achieving genetic reshuffling and genome repair. To maintain genomic integrity, specialized resolvase enzymes cleave the entangled DNA into two discrete DNA molecules. However, it is unclear how two similar stacking isomers are distinguished, and how a cognate sequence is found and recognized to achieve accurate recombination. We here use single-molecule fluorescence observation and cluster analysis to examine how prototypic bacterial resolvase RuvC singles out two of the four HJ strands and achieves sequence-specific cleavage. We find that RuvC first exploits, then constrains the dynamics of intrinsic HJ isomer exchange at a sampled branch position to direct cleavage toward the catalytically competent HJ conformation and sequence, thus controlling recombination output at minimal energetic cost. Our model of rapid DNA scanning followed by ‘snap-locking’ of a cognate sequence is strikingly consistent with the conformational proofreading of other DNA-modifying enzymes.

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