Browsing by Author "Durand, Neva C."
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Item A rapid, low-cost, and highly sensitive SARS-CoV-2 diagnostic based on whole-genome sequencing(Public Library of Science, 2023) Adastra, Per A.; Durand, Neva C.; Mitra, Namita; Pulido, Saul Godinez; Mahajan, Ragini; Blackburn, Alyssa; Colaric, Zane L.; Theisen, Joshua W. M.; Weisz, David; Dudchenko, Olga; Gnirke, Andreas; Rao, Suhas S. P.; Kaur, Parwinder; Aiden, Erez Lieberman; Aiden, Aviva Presser; Center for Theoretical Biological PhysicsEarly detection of SARS-CoV-2 infection is key to managing the current global pandemic, as evidence shows the virus is most contagious on or before symptom onset. Here, we introduce a low-cost, high-throughput method for diagnosing and studying SARS-CoV-2 infection. Dubbed Pathogen-Oriented Low-Cost Assembly & Re-Sequencing (POLAR), this method amplifies the entirety of the SARS-CoV-2 genome. This contrasts with typical RT-PCR-based diagnostic tests, which amplify only a few loci. To achieve this goal, we combine a SARS-CoV-2 enrichment method developed by the ARTIC Network (https://artic.network/) with short-read DNA sequencing and de novo genome assembly. Using this method, we can reliably (>95% accuracy) detect SARS-CoV-2 at a concentration of 84 genome equivalents per milliliter (GE/mL). The vast majority of diagnostic methods meeting our analytical criteria that are currently authorized for use by the United States Food and Drug Administration with the Coronavirus Disease 2019 (COVID-19) Emergency Use Authorization require higher concentrations of the virus to achieve this degree of sensitivity and specificity. In addition, we can reliably assemble the SARS-CoV-2 genome in the sample, often with no gaps and perfect accuracy given sufficient viral load. The genotypic data in these genome assemblies enable the more effective analysis of disease spread than is possible with an ordinary binary diagnostic. These data can also help identify vaccine and drug targets. Finally, we show that the diagnoses obtained using POLAR of positive and negative clinical nasal mid-turbinate swab samples 100% match those obtained in a clinical diagnostic lab using the Center for Disease Control’s 2019-Novel Coronavirus test. Using POLAR, a single person can manually process 192 samples over an 8-hour experiment at the cost of ~$36 per patient (as of December 7th, 2022), enabling a 24-hour turnaround with sequencing and data analysis time. We anticipate that further testing and refinement will allow greater sensitivity using this approach.Item Chromatin architecture transitions from zebrafish sperm through early embryogenesis(Cold Spring Harbor Laboratory Press, 2021) Wike, Candice L.; Guo, Yixuan; Tan, Mengyao; Nakamura, Ryohei; Shaw, Dana Klatt; Díaz, Noelia; Whittaker-Tademy, Aneasha F.; Durand, Neva C.; Aiden, Erez Lieberman; Vaquerizas, Juan M.; Grunwald, David; Takeda, Hiroyuki; Cairns, Bradley R.; Center for Theoretical Biological PhysicsChromatin architecture mapping in 3D formats has increased our understanding of how regulatory sequences and gene expression are connected and regulated in a genome. The 3D chromatin genome shows extensive remodeling during embryonic development, and although the cleavage-stage embryos of most species lack structure before zygotic genome activation (pre-ZGA), zebrafish has been reported to have structure. Here, we aimed to determine the chromosomal architecture in paternal/sperm zebrafish gamete cells to discern whether it either resembles or informs early pre-ZGA zebrafish embryo chromatin architecture. First, we assessed the higher-order architecture through advanced low-cell in situ Hi-C. The structure of zebrafish sperm, packaged by histones, lacks topological associated domains and instead displays “hinge-like” domains of ∼150 kb that repeat every 1–2 Mbs, suggesting a condensed repeating structure resembling mitotic chromosomes. The pre-ZGA embryos lacked chromosomal structure, in contrast to prior work, and only developed structure post-ZGA. During post-ZGA, we find chromatin architecture beginning to form at small contact domains of a median length of ∼90 kb. These small contact domains are established at enhancers, including super-enhancers, and chemical inhibition of Ep300a (p300) and Crebbpa (CBP) activity, lowering histone H3K27ac, but not transcription inhibition, diminishes these contacts. Together, this study reveals hinge-like domains in histone-packaged zebrafish sperm chromatin and determines that the initial formation of high-order chromatin architecture in zebrafish embryos occurs after ZGA primarily at enhancers bearing high H3K27ac.Item CTCF looping is established during gastrulation in medaka embryos(Cold Spring Harbor Laboratory Press, 2021) Nakamura, Ryohei; Motai, Yuichi; Kumagai, Masahiko; Wike, Candice L.; Nishiyama, Haruyo; Nakatani, Yoichiro; Durand, Neva C.; Kondo, Kaori; Kondo, Takashi; Tsukahara, Tatsuya; Shimada, Atsuko; Cairns, Bradley R.; Aiden, Erez Lieberman; Morishita, Shinichi; Takeda, Hiroyuki; Center for Theoretical Biological PhysicsChromatin looping plays an important role in genome regulation. However, because ChIP-seq and loop-resolution Hi-C (DNA-DNA proximity ligation) are extremely challenging in mammalian early embryos, the developmental stage at which cohesin-mediated loops form remains unknown. Here, we study early development in medaka (the Japanese killifish, Oryzias latipes) at 12 time points before, during, and after gastrulation (the onset of cell differentiation) and characterize transcription, protein binding, and genome architecture. We find that gastrulation is associated with drastic changes in genome architecture, including the formation of the first loops between sites bound by the insulator protein CTCF and a large increase in the size of contact domains. In contrast, the binding of the CTCF is fixed throughout embryogenesis. Loops form long after genome-wide transcriptional activation, and long after domain formation seen in mouse embryos. These results suggest that, although loops may play a role in differentiation, they are not required for zygotic transcription. When we repeated our experiments in zebrafish, loops did not emerge until gastrulation, that is, well after zygotic genome activation. We observe that loop positions are highly conserved in synteny blocks of medaka and zebrafish, indicating that the 3D genome architecture has been maintained for >110–200 million years of evolution.Item Walking along chromosomes with super-resolution imaging, contact maps, and integrative modeling(Public Library of Science, 2018) Nir, Guy; Farabella, Irene; Estrada, Cynthia Pérez; Ebeling, Carl G.; Beliveau, Brian J.; Sasaki, Hiroshi M.; Lee, S. Dean; Nguyen, Son C.; McCole, Ruth B.; Chattoraj, Shyamtanu; Erceg, Jelena; Abed, Jumana AlHaj; Martins, Nuno M.C.; Nguyen, Huy Q.; Hannan, Mohammed A.; Russell, Sheikh; Durand, Neva C.; Rao, Suhas S.P.; Kishi, Jocelyn Y.; Soler-Vila, Paula; Pierro, Michele Di; Onuchic, José N.; Callahan, Steven P.; Schreiner, John M.; Stuckey, Jeff A.; Yin, Peng; Aiden, Erez Lieberman; Marti-Renom, Marc A.; Wu, C.-tingChromosome organization is crucial for genome function. 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. Using the super-resolution microscopy methods of OligoSTORM and OligoDNA-PAINT, we trace 8 megabases of human chromosome 19, visualizing structures ranging in size from a few kilobases to over a megabase. Focusing on chromosomal regions that contribute to compartments, we discover distinct structures that, in spite of considerable variability, can predict whether such regions correspond to active (A-type) or inactive (B-type) compartments. Imaging through the depths of entire nuclei, we capture pairs of homologous regions in diploid cells, obtaining evidence that maternal and paternal homologous regions can be differentially organized. Finally, using restraint-based modeling to integrate imaging and Hi-C data, we implement a method-integrative modeling of genomic regions (IMGR)-to increase the genomic resolution of our traces to 10 kb.