scholarly journals Super resolution imaging of a distinct chromatin loop in human lymphoblastoid cells

2019 ◽  
Author(s):  
Jacqueline Jufen Zhu ◽  
Zofia Parteka ◽  
Byoungkoo Lee ◽  
Przemyslaw Szalaj ◽  
Ping Wang ◽  
...  
Keyword(s):  
Single Molecule ◽  
Genome Structure ◽  
Three Dimensional ◽  
Super Resolution ◽  
Chromatin Loop ◽  
Imaging Data ◽  
Sequencing Data ◽  
Cellular Functions ◽  
Sequencing Technologies ◽  
Chromatin Folding

AbstractThe three-dimensional genome structure plays a fundamental role in gene regulation and cellular functions. Recent studies in genomics based on sequencing technologies inferred the very basic functional chromatin folding structures of the genome known as chromatin loops, the long-range chromatin interactions that are often mediated by protein factors. To visualize the looping structure of chromatin we applied super-resolution microscopy iPALM to image a specific chromatin loop in GM12878 cells. Totally, we have generated six images of the target chromatin region at the single molecule resolution. To infer the chromatin structures from the captured images, we modeled them as looping conformations using different computational algorithms and then evaluated the models by comparing with Hi-C data to examine the concordance. The results showed a good correlation between the imaging data and sequencing data, suggesting the visualization of higher-order chromatin structures for the very short genomic segments can be realized by microscopic imaging.

scholarly journals Walking along chromosomes with super-resolution imaging, contact maps, and integrative modeling

2018 ◽  
Author(s):  
Guy Nir ◽  
Irene Farabella ◽  
Cynthia Pérez Estrada ◽  
Carl G. Ebeling ◽  
Brian J. Beliveau ◽  
...  
Keyword(s):  
Single Molecule ◽  
Three Dimensional ◽  
Super Resolution ◽  
Imaging Data ◽  
Chromosomal Dna ◽  
Contact Maps ◽  
Integrative Modeling ◽  
Resolution Imaging ◽  
Three Dimensional Models ◽  
Single Molecule Localization Microscopy

AbstractChromosome structure is thought to be crucial for proper functioning of the nucleus. Here, we present a method for visualizing chromosomal DNA at super-resolution and then integrating Hi-C data to produce three-dimensional models of chromosome organization. We begin by applying Oligopaint probes and the single-molecule localization microscopy methods of OligoSTORM and OligoDNA-PAINT to image 8 megabases of human chromosome 19, discovering that chromosomal regions contributing to compartments can form distinct structures. Intriguingly, our data also suggest that homologous maternal and paternal regions may be differentially organized. Finally, we integrate imaging data with Hi-C and restraint-based modeling using a method called integrative modeling of genomic regions (IMGR) to increase the genomic resolution of our traces to 10 kb.One Sentence SummarySuper-resolution genome tracing, contact maps, and integrative modeling enable 10 kb resolution glimpses of chromosome folding.

scholarly journals Sequoia: an interactive visual analytics platform for interpretation and feature extraction from nanopore sequencing datasets

BMC Genomics ◽  
2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Ratanond Koonchanok ◽  
Swapna Vidhur Daulatabad ◽  
Quoseena Mir ◽  
Khairi Reda ◽  
Sarath Chandra Janga
Keyword(s):  
Single Molecule ◽  
Visual Analytics ◽  
Visual Analysis ◽  
Direct Sequencing ◽  
Visual Exploration ◽  
Nanopore Sequencing ◽  
Sequencing Data ◽  
Rna Sequences ◽  
Sequencing Technologies ◽  
Signal Features

Abstract Background Direct-sequencing technologies, such as Oxford Nanopore’s, are delivering long RNA reads with great efficacy and convenience. These technologies afford an ability to detect post-transcriptional modifications at a single-molecule resolution, promising new insights into the functional roles of RNA. However, realizing this potential requires new tools to analyze and explore this type of data. Result Here, we present Sequoia, a visual analytics tool that allows users to interactively explore nanopore sequences. Sequoia combines a Python-based backend with a multi-view visualization interface, enabling users to import raw nanopore sequencing data in a Fast5 format, cluster sequences based on electric-current similarities, and drill-down onto signals to identify properties of interest. We demonstrate the application of Sequoia by generating and analyzing ~ 500k reads from direct RNA sequencing data of human HeLa cell line. We focus on comparing signal features from m6A and m5C RNA modifications as the first step towards building automated classifiers. We show how, through iterative visual exploration and tuning of dimensionality reduction parameters, we can separate modified RNA sequences from their unmodified counterparts. We also document new, qualitative signal signatures that characterize these modifications from otherwise normal RNA bases, which we were able to discover from the visualization. Conclusions Sequoia’s interactive features complement existing computational approaches in nanopore-based RNA workflows. The insights gleaned through visual analysis should help users in developing rationales, hypotheses, and insights into the dynamic nature of RNA. Sequoia is available at https://github.com/dnonatar/Sequoia.

scholarly journals Quantifying accuracy and heterogeneity in single-molecule super-resolution microscopy

2019 ◽  
Author(s):  
Hesam Mazidi ◽  
Tianben Ding ◽  
Arye Nehorai ◽  
Matthew D. Lew
Keyword(s):  
Single Molecule ◽  
Computational Models ◽  
Amyloid Fibrils ◽  
Imaging System ◽  
Simulated Data ◽  
Super Resolution ◽  
Ground Truth ◽  
Localization Algorithm ◽  
Reconstruction Algorithms ◽  
Imaging Data

The resolution and accuracy of single-molecule localization micro-scopes (SMLMs) are routinely benchmarked using simulated data, calibration “rulers,” or comparisons to secondary imaging modalities. However, these methods cannot quantify the nanoscale accuracy of an arbitrary SMLM dataset. Here, we show that by computing localization stability under a well-chosen perturbation with accurate knowledge of the imaging system, we can robustly measure the confidence of individual localizations without ground-truth knowledge of the sample. We demonstrate that our method, termed Wasserstein-induced flux (WIF), measures the accuracy of various reconstruction algorithms directly on experimental 2D and 3D data of microtubules and amyloid fibrils. We further show that WIF confidences can be used to evaluate the mismatch between computational models and imaging data, enhance the accuracy and resolution of recon-structed structures, and discover hidden molecular heterogeneities. As a computational methodology, WIF is broadly applicable to any SMLM dataset, imaging system, and localization algorithm.

scholarly journals A Transposon Story: From TE Content to TE Dynamic Invasion of Drosophila Genomes Using the Single-Molecule Sequencing Technology from Oxford Nanopore

Cells ◽  
2020 ◽  
Vol 9 (8) ◽  
pp. 1776
Author(s):  
Mourdas Mohamed ◽  
Nguyet Thi-Minh Dang ◽  
Yuki Ogyama ◽  
Nelly Burlet ◽  
Bruno Mugat ◽  
...  
Keyword(s):  
Single Molecule ◽  
Wild Type ◽  
Sequencing Data ◽  
Short Read ◽  
Short Read Sequencing ◽  
Sequencing Technologies ◽  
Oxford Nanopore ◽  
In The Wild ◽  
Successive Generations ◽  
Type Strains

Transposable elements (TEs) are the main components of genomes. However, due to their repetitive nature, they are very difficult to study using data obtained with short-read sequencing technologies. Here, we describe an efficient pipeline to accurately recover TE insertion (TEI) sites and sequences from long reads obtained by Oxford Nanopore Technology (ONT) sequencing. With this pipeline, we could precisely describe the landscapes of the most recent TEIs in wild-type strains of Drosophila melanogaster and Drosophila simulans. Their comparison suggests that this subset of TE sequences is more similar than previously thought in these two species. The chromosome assemblies obtained using this pipeline also allowed recovering piRNA cluster sequences, which was impossible using short-read sequencing. Finally, we used our pipeline to analyze ONT sequencing data from a D. melanogaster unstable line in which LTR transposition was derepressed for 73 successive generations. We could rely on single reads to identify new insertions with intact target site duplications. Moreover, the detailed analysis of TEIs in the wild-type strains and the unstable line did not support the trap model claiming that piRNA clusters are hotspots of TE insertions.

scholarly journals 3D tracking of extracellular vesicles by holographic fluorescence imaging

2020 ◽  
Vol 6 (45) ◽  
pp. eabc2508
Author(s):  
Matz Liebel ◽  
Jaime Ortega Arroyo ◽  
Vanesa Sanz Beltrán ◽  
Johann Osmond ◽  
Ala Jo ◽  
...  
Keyword(s):  
Extracellular Vesicles ◽  
Single Molecule ◽  
Three Dimensional ◽  
Super Resolution ◽  
Fluorescent Detection ◽  
Live Cells ◽  
Fluorescent Light ◽  
3D Tracking ◽  
Anisotropic Motion ◽  
Lag Times

Fluorescence microscopy is the method of choice in biology for its molecular specificity and super-resolution capabilities. However, it is limited to a narrow z range around one observation plane. Here, we report an imaging approach that recovers the full electric field of fluorescent light with single-molecule sensitivity. We expand the principle of digital holography to fast fluorescent detection by eliminating the need for phase cycling and enable three-dimensional (3D) tracking of individual nanoparticles with an in-plane resolution of 15 nm and a z-range of 8 mm. As a proof-of-concept biological application, we image the 3D motion of extracellular vesicles (EVs) inside live cells. At short time scales (<4 s), we resolve near-isotropic 3D diffusion and directional transport. For longer lag times, we observe a transition toward anisotropic motion with the EVs being transported over long distances in the axial plane while being confined in the horizontal dimension.

scholarly journals PSF shaping using adaptive optics for three-dimensional single-molecule super-resolution imaging and tracking

2012 ◽  
Vol 20 (5) ◽  
pp. 4957 ◽  
Author(s):  
Ignacio Izeddin ◽  
Mohamed El Beheiry ◽  
Jordi Andilla ◽  
Daniel Ciepielewski ◽  
Xavier Darzacq ◽  
...  
Keyword(s):  
Adaptive Optics ◽  
Single Molecule ◽  
Three Dimensional ◽  
Super Resolution ◽  
Resolution Imaging

scholarly journals Obtaining 3D Super-resolution Information from 2D Super-resolution Images through a 2D-to-3D Transformation Algorithm

2017 ◽  
Author(s):  
Andrew Ruba ◽  
Wangxi Luo ◽  
Joseph Kelich ◽  
Weidong Yang
Keyword(s):  
Single Molecule ◽  
Rotational Symmetry ◽  
Three Dimensional ◽  
Super Resolution ◽  
Live Cells ◽  
Limited Information ◽  
Spatiotemporal Resolution ◽  
Superresolution Microscopy ◽  
Localization Analysis ◽  
Probability Information

AbstractCurrently, it is highly desirable but still challenging to obtain three-dimensional (3D) superresolution information of structures in fixed specimens as well as dynamic processes in live cells with a high spatiotemporal resolution. Here we introduce an approach, without using 3D superresolution microscopy or real-time 3D particle tracking, to achieve 3D sub-diffraction-limited information with a spatial resolution of ≤ 1 nm. This is a post-localization analysis that transforms 2D super-resolution images or 2D single-molecule localization distributions into their corresponding 3D spatial probability information. The method has been successfully applied to obtain structural and functional information for 25-300 nm sub-cellular organelles that have rotational symmetry. In this article, we will provide a comprehensive analysis of this method by using experimental data and computational simulations.

scholarly journals Photoregulated Fluxional Fluorophores for Live-Cell Super-Resolution Microscopy with No Apparent Photobleaching

2019 ◽  
Author(s):  
Elias A. Halabi ◽  
Dorothea Pinotsi ◽  
Pablo Rivera-Fuentes
Keyword(s):  
Single Molecule ◽  
Three Dimensional ◽  
Super Resolution ◽  
Time Lapse ◽  
Switching Behavior ◽  
Spatiotemporal Resolution ◽  
New Information ◽  
Human Neurons ◽  
Long Time ◽  
Intracellular Organelles

Photoswitchable molecules have found multiple applications in the physical and life sciences because their properties can be modulated with light. Fluxional molecules, which undergo rapid degenerate rearrangements in the electronic ground state, also exhibit switching behavior. The stochastic nature of fluxional switching, however, has hampered its application in the development of functional molecules and materials. Here we combine photoswitching and fluxionality to develop a fluorophore that enables very long (>30 min) time-lapse single-molecule localization microscopy in living cells with minimal phototoxicity and no apparent photobleaching. These long time-lapse experiments allowed us to track intracellular organelles with unprecedented spatiotemporal resolution, revealing new information of the three-dimensional compartmentalization of synaptic vesicle trafficking in live human neurons.

Critical assessment of bioinformatics methods for the characterization of pathological repeat expansions with single-molecule sequencing data

2019 ◽  
Vol 21 (6) ◽  
pp. 1971-1986 ◽  
Author(s):  
Matteo Chiara ◽  
Federico Zambelli ◽  
Ernesto Picardi ◽  
David S Horner ◽  
Graziano Pesole
Keyword(s):  
Single Molecule ◽  
Tandem Repeats ◽  
Simulated Data ◽  
Detailed Comparison ◽  
Sequencing Data ◽  
Single Molecule Sequencing ◽  
Sequencing Technologies ◽  
Repeat Expansions

Abstract A number of studies have reported the successful application of single-molecule sequencing technologies to the determination of the size and sequence of pathological expanded microsatellite repeats over the last 5 years. However, different custom bioinformatics pipelines were employed in each study, preventing meaningful comparisons and somewhat limiting the reproducibility of the results. In this review, we provide a brief summary of state-of-the-art methods for the characterization of expanded repeats alleles, along with a detailed comparison of bioinformatics tools for the determination of repeat length and sequence, using both real and simulated data. Our reanalysis of publicly available human genome sequencing data suggests a modest, but statistically significant, increase of the error rate of single-molecule sequencing technologies at genomic regions containing short tandem repeats. However, we observe that all the methods herein tested, irrespective of the strategy used for the analysis of the data (either based on the alignment or assembly of the reads), show high levels of sensitivity in both the detection of expanded tandem repeats and the estimation of the expansion size, suggesting that approaches based on single-molecule sequencing technologies are highly effective for the detection and quantification of tandem repeat expansions and contractions.

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