Littérature scientifique sur le sujet « Chromatin sequencing »
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Articles de revues sur le sujet "Chromatin sequencing"
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Soleimani, Vahab D., Gareth A. Palidwor, Parameswaran Ramachandran, Theodore J. Perkins et Michael A. Rudnicki. « Chromatin tandem affinity purification sequencing ». Nature Protocols 8, no 8 (11 juillet 2013) : 1525–34. http://dx.doi.org/10.1038/nprot.2013.088.
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Jukam, David, Charles Limouse, Owen K. Smith, Viviana I. Risca, Jason C. Bell et Aaron F. Straight. « Chromatin‐Associated RNA Sequencing (ChAR‐seq) ». Current Protocols in Molecular Biology 126, no 1 (20 février 2019) : e87. http://dx.doi.org/10.1002/cpmb.87.
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Stergachis, Andrew B., Brian M. Debo, Eric Haugen, L. Stirling Churchman et John A. Stamatoyannopoulos. « Single-molecule regulatory architectures captured by chromatin fiber sequencing ». Science 368, no 6498 (25 juin 2020) : 1449–54. http://dx.doi.org/10.1126/science.aaz1646.
Texte intégralRésumé :
Gene regulation is chiefly determined at the level of individual linear chromatin molecules, yet our current understanding of cis-regulatory architectures derives from fragmented sampling of large numbers of disparate molecules. We developed an approach for precisely stenciling the structure of individual chromatin fibers onto their composite DNA templates using nonspecific DNA N6-adenine methyltransferases. Single-molecule long-read sequencing of chromatin stencils enabled nucleotide-resolution readout of the primary architecture of multikilobase chromatin fibers (Fiber-seq). Fiber-seq exposed widespread plasticity in the linear organization of individual chromatin fibers and illuminated principles guiding regulatory DNA actuation, the coordinated actuation of neighboring regulatory elements, single-molecule nucleosome positioning, and single-molecule transcription factor occupancy. Our approach and results open new vistas on the primary architecture of gene regulation.
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Xie, Wenhui, Yilang Ke, Qinyi You, Jing Li, Lu Chen, Dang Li, Jun Fang et al. « Single-Cell RNA Sequencing and Assay for Transposase-Accessible Chromatin Using Sequencing Reveals Cellular and Molecular Dynamics of Aortic Aging in Mice ». Arteriosclerosis, Thrombosis, and Vascular Biology 42, no 2 (février 2022) : 156–71. http://dx.doi.org/10.1161/atvbaha.121.316883.
Texte intégralRésumé :
Objective: The impact of vascular aging on cardiovascular diseases has been extensively studied; however, little is known regarding the cellular and molecular mechanisms underlying age-related vascular aging in aortic cellular subpopulations. Approach and Results: Transcriptomes and transposase-accessible chromatin profiles from the aortas of 4-, 26-, and 86-week-old C57/BL6J mice were analyzed using single-cell RNA sequencing and assay for transposase-accessible chromatin sequencing. By integrating the heterogeneous transcriptome and chromatin accessibility data, we identified cell-specific TF (transcription factor) regulatory networks and open chromatin states. We also determined that aortic aging affects cell interactions, inflammation, cell type composition, dysregulation of transcriptional control, and chromatin accessibility. Endothelial cells 1 have higher gene set activity related to cellular senescence and aging than do endothelial cells 2. Moreover, construction of senescence trajectories shows that endothelial cell 1 and fibroblast senescence is associated with distinct TF open chromatin states and an mRNA expression model. Conclusions: Our data provide a system-wide model for transcriptional and epigenetic regulation during aortic aging at single-cell resolution.
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Wu, Weixin, Zhangming Yan, Tri C. Nguyen, Zhen Bouman Chen, Shu Chien et Sheng Zhong. « Mapping RNA–chromatin interactions by sequencing with iMARGI ». Nature Protocols 14, no 11 (16 octobre 2019) : 3243–72. http://dx.doi.org/10.1038/s41596-019-0229-4.
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Gorkin, David U., Iros Barozzi, Yuan Zhao, Yanxiao Zhang, Hui Huang, Ah Young Lee, Bin Li et al. « An atlas of dynamic chromatin landscapes in mouse fetal development ». Nature 583, no 7818 (29 juillet 2020) : 744–51. http://dx.doi.org/10.1038/s41586-020-2093-3.
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AbstractThe Encyclopedia of DNA Elements (ENCODE) project has established a genomic resource for mammalian development, profiling a diverse panel of mouse tissues at 8 developmental stages from 10.5 days after conception until birth, including transcriptomes, methylomes and chromatin states. Here we systematically examined the state and accessibility of chromatin in the developing mouse fetus. In total we performed 1,128 chromatin immunoprecipitation with sequencing (ChIP–seq) assays for histone modifications and 132 assay for transposase-accessible chromatin using sequencing (ATAC–seq) assays for chromatin accessibility across 72 distinct tissue-stages. We used integrative analysis to develop a unified set of chromatin state annotations, infer the identities of dynamic enhancers and key transcriptional regulators, and characterize the relationship between chromatin state and accessibility during developmental gene regulation. We also leveraged these data to link enhancers to putative target genes and demonstrate tissue-specific enrichments of sequence variants associated with disease in humans. The mouse ENCODE data sets provide a compendium of resources for biomedical researchers and achieve, to our knowledge, the most comprehensive view of chromatin dynamics during mammalian fetal development to date.
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Jahan, Sanzida, Tasnim H. Beacon, Wayne Xu et James R. Davie. « Atypical chromatin structure of immune-related genes expressed in chicken erythrocytes ». Biochemistry and Cell Biology 98, no 2 (avril 2020) : 171–77. http://dx.doi.org/10.1139/bcb-2019-0107.
Texte intégralRésumé :
The major biological role of red blood cells is to carry oxygen to the tissues in the body. However, another role of the erythroid cell is to participate in the immune response. Mature erythrocytes from chickens express Toll-like receptors and several cytokines in response to stimulation of the immune system. We previously reported the application of a biochemical fractionation protocol to isolate highly enriched transcribed DNA from polychromatic erythrocytes from chickens. In conjunction with next-generation DNA, RNA sequencing, chromatin immunoprecipitation-DNA sequencing, and formaldehyde-assisted isolation of regulatory elements (FAIRE) sequencing, we identified the active chromosomal compartments and determined their structural signatures in relation to expression levels. Here, we present the detailed chromatin characteristics of erythroid genes participating in the innate immune response. Our studies revealed an atypical chromatin structure for several genes coding for Toll-like receptors, interleukins, and interferon regulatory factors. The body of these genes had nucleosome-free regions intermingled with nucleosomes modified with H3K4me3 and H3K27ac, suggesting a dynamic unstable chromatin structure. We further show that human genes involved in cell identity have gene bodies with the same chromatin-instability features as the chicken polychromatic erythrocyte genes participating in the innate immune response.
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Guo, Ziwei, Xinhong Liu et Mo Chen. « Defining pervasive transcription units using chromatin RNA-sequencing data ». STAR Protocols 3, no 2 (juin 2022) : 101442. http://dx.doi.org/10.1016/j.xpro.2022.101442.
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Vega, Vinsensius B., Edwin Cheung, Nallasivam Palanisamy et Wing-Kin Sung. « Inherent Signals in Sequencing-Based Chromatin-ImmunoPrecipitation Control Libraries ». PLoS ONE 4, no 4 (15 avril 2009) : e5241. http://dx.doi.org/10.1371/journal.pone.0005241.
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Bright, Ann Rose, et Gert Jan C. Veenstra. « Assay for Transposase-Accessible Chromatin-Sequencing Using Xenopus Embryos ». Cold Spring Harbor Protocols 2019, no 1 (24 juillet 2018) : pdb.prot098327. http://dx.doi.org/10.1101/pdb.prot098327.
Texte intégralThèses sur le sujet "Chromatin sequencing"
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Cook, David. « SNF2H-Mediated Chromatin Remodelling and Its Regulation of the Pluripotent State ». Thesis, Université d'Ottawa / University of Ottawa, 2016. http://hdl.handle.net/10393/35097.
Texte intégralRésumé :
In embryonic stem cells (ESCs), the SWI/SNF, CHD, and INO80 families of ATP-dependent chromatin remodellers have been implicated in maintaining pluripotency-associated gene expression, however the involvement of ISWI family remodellers has yet to be defined. Here, we explore the importance of the mammalian ISWI homologue SNF2H (Smarca5) by deriving a conditional knockout mouse ESC line and observing the consequences of SNF2H depletion on the pluripotent state. Cre-mediated deletion of Snf2h disrupts hallmark characteristics of pluripotency, resulting in distinct morphological changes; reduced expression of the master transcription factors Oct4, Sox2, and Nanog; and reduced alkaline phosphatase activity.
To understand the mechanisms of SNF2H-mediated regulation, we mapped SNF2H-bound nucleosomes genome-wide. SNF2H is broadly distributed across the genome, but is preferentially enriched at active regulatory regions and transcription factor binding sites. These findings demonstrate the importance of SNF2H in ESCs and shed light on genome-wide mechanisms of transcriptional regulation.
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LUCINI, FEDERICA. « Unconventional nuclear architecture in CD4+ T lymphocytes uncouples chromatin solubility from function ». Doctoral thesis, Università degli Studi di Milano-Bicocca, 2020. http://hdl.handle.net/10281/262913.
Texte intégralRésumé :
Nei nuclei delle cellule eucarioti, l'informazione genetica codificata nel DNA è concentrata nel microscopico volume nucleare in forma di cromatina, un complesso di DNA e proteine. I meccanismi molecolari che gestiscono la compattazione e il ripiegamento della cromatina e che consentono l'espressione mirata delle porzioni di genoma necessarie alle attività della cellula sono noti come ‘epigenoma’. L’azione dell’epigenoma determina un avvolgimento e un posizionamento nucleare della cromatina specifico per ogni tipo cellulare, con aree dense e trascrizionalmente inattive (eterocromatina) ed aree meno dense, ricche di geni e trascrizionalmente attive (eucromatina).
In questo nostro lavoro descriviamo l'organizzazione della cromatina nel nucleo di diverse popolazioni cellulari e ne analizziamo alcuni aspetti fisiologici e patologici.
Innanzitutto, studiando le cellule staminali muscolari di topi privi della proteina strutturale nucleare lamina-A/C, descriviamo un irregolare processo di differenziamento dovuto alla redistribuzione dei repressori trascrizionali del gruppo Polycomb (PcG proteins), che dai i loro geni target si diffondono verso regioni cromatiniche fiancheggianti. La conseguente alterazione nell’espressione genica causa l’esaurimento prematuro della riserva di cellule staminali quiescenti e l’accumulo di grasso intramuscolare, portando a una senescenza accelerata e alla distrofia muscolare.
D’altro canto, anche il progressivo accumulo di una forma aberrante di lamina-A, la progerina, caratteristica della sindrome di Hutchinson-Gilford (HGPS), causa gravi alterazioni nella struttura della cromatina. In particolare, la progerina interferisce con le strutture eterocromatiniche periferiche associate alla lamina nucleare, i Lamina Associated Domains (LADs). Per il nostro secondo progetto abbiamo sviluppato un nuovo metodo, SAMMY-seq, basato sull’high-throughput sequencing di frazioni di cromatina con diversa solubilità. Tramite questa tecnologia, individuiamo alterazioni nella solubilità dell’eterocromatina in fibroblasti primari derivanti da pazienti progerici in uno stadio precoce di malattia. I cambiamenti strutturali osservati a questo stadio non alterano la deposizione del marcatore eterocromatinico H3K9me3, ma sono associati a variazioni sito-specifiche nella regolazione trascrizionale di geni target delle PcG proteins.
Infine, ottimizzando ulteriormente il protocollo di SAMMY-seq, nel nostro terzo progetto mostriamo un’organizzazione non convenzionale della cromatina nei linfociti T CD4+ quiescenti derivanti da sangue periferico di donatori. In queste cellule, l’eterocromatina risulta sensibile alla digestione enzimatica operata dalla DNAsi, mentre l’eucromatina si rivela resistente a diversi processi di estrazione. Un’analisi preliminare del contenuto di questi compartimenti indica la presenza, nell’eucromatina, dei geni specifici per l’attivazione linfocitaria, oltre che dei geni attivi. Ulteriori studi chiariranno il ruolo di questa organizzazione non convenzionale della cromatina nella funzione cellulare linfocitaria.In every eukaryotic cell, the genomic information coded in the DNA is packed into the small nuclear volume as chromatin, a complex of DNA and proteins. The ensemble of molecular mechanisms that organize chromatin compaction and allow the specific expression of the portions of genome useful for cell’s biological functions is known as the epigenome. As a result of epigenome activity, chromatin is folded and positioned in the nucleus in a cell-specific manner, generating areas of highly compacted, repressed, heterochromatin and areas of decondensed, gene-rich and transcriptionally active, euchromatin. In our work, we describe chromatin organization in different cell populations and analyse some of its implications in the physiological functions and pathological dysfunctions of the cell. In the first project, we focus on murine muscle stem cells lacking the nuclear structural protein Lamin A/C. We show their irregular differentiation program, due to a spreading of Polycomb group (PcG) of proteins repressors from their target genes over the flanking regions. The consequent alteration in gene expression cause premature exhaustion of quiescent stem cells and accumulation of intramuscular fat, resulting in accelerated senescence and muscular dystrophy progression. On the other hand, the progressive accumulation of a Lamin A aberrant form, Progerin, in Hutchinson-Gilford progeria syndrome (HGPS) also leads to chromatin structure disruption. In particular, it interferes with Lamina Associated Domains (LADs), the peripheral heterochromatin structures associated to the nuclear lamina. For our second project, we develop a new method, SAMMY-seq, based on high-throughput sequencing of chromatin fractions of different solubility. Thanks to this technology, we highlight early changes in heterochromatin accessibility in human HGPS primary fibroblasts. This early structural changes do not alter the deposition of the H3K9me3 heterochromatin mark but are associated with site-specific variations in the PcG-dependent transcriptional regulation. Finally, further improving SAMMY-seq technology, in our third project we describe an unconventional genome organization in resting human CD4+ T lymphocytes extracted from the peripheral blood of healthy donors. In these cells, heterochromatin is sensitive to DNAse digestion while euchromatin is resistant to serial processes of extraction. Preliminary analysis of the content of these compartments suggests that euchromatin contains, beside the actively transcribed genes, also inactive genes specific for lymphocyte activation. Further studies will elucidate the role of this unconventional chromatin organization in lymphocytes functions.
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Aitken, Sarah Jane. « The pathological and genomic impact of CTCF depletion in mammalian model systems ». Thesis, University of Cambridge, 2018. https://www.repository.cam.ac.uk/handle/1810/284403.
Texte intégralRésumé :
CCCTC-binding factor (CTCF) binds DNA, thereby helping to partition the mammalian genome into discrete structural and regulatory domains. In doing so, it insulates chromatin and fine-tunes gene activation, repression, and silencing. Complete removal of CTCF from mammalian cells causes catastrophic genomic dysregulation, most likely due to widespread collapse of 3D chromatin looping within the nucleus. In contrast, Ctcf hemizygous mice with lifelong reduction in CTCF expression are viable but have an increased incidence of spontaneous multi-lineage malignancies. In addition, CTCF is mutated in many human cancers and is thus implicated as a tumour suppressor gene. This study aimed to interrogate the genome-wide consequences of a reduced genomic concentration of Ctcf and its implications for carcinogenesis. In a genetically engineered mouse model, Ctcf hemizygous cells showed modest but robust changes in almost a thousand sites of genomic CTCF occupancy; these were enriched for lower affinity binding events with weaker evolutionary conservation across the mouse lineage. Furthermore, several hundred genes concentrated in cancer-related pathways were dysregulated due to changes in transcriptional regulation. Global chromatin structure was preserved but some loop interactions were destabilised, often around differentially expressed genes and their enhancers. Importantly, these transcriptional alterations were also seen in human cancers. These findings were then examined in a hepatocyte-specific mouse model of Ctcf hemizygosity with diethylnitrosamine-induced liver tumours. Ctcf hemizygous mice had a subtle liver-specific phenotype, although the overall tumour burden in Ctcf hemizygous and wild-type mice was the same. Using whole genome sequencing, the highly reproducible mutational signature caused by DEN exposure was characterised, revealing that Braf(V637E), orthologous to BRAF(V600E) in humans, was the predominant oncogenic driver in these liver tumours. Taken together, while Ctcf loss is partially physiologically compensated, chronic CTCF depletion dysregulates gene expression by subtly altering transcriptional regulation. This study also represents the first comprehensive genome-wide and histopathological characterisation of this commonly used liver cancer model.
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Deng, Chengyu. « Microfluidics for Low Input Epigenomic Analysis and Its Application to Brain Neuroscience ». Diss., Virginia Tech, 2021. http://hdl.handle.net/10919/101765.
Texte intégralRésumé :
The epigenome carries dynamic information that controls gene expression and maintains cell identity during both disease and normal development. The inherent plasticity of the epigenome paves new avenues for developing diagnostic and therapeutic tools for human diseases. Microfluidic technology has improved the sensitivity and resolution of epigenomic analysis due to its outstanding ability to manipulate nanoliter-scale liquid volumes. In this thesis, I report three projects focusing on low-input, cell-type-specific and spatially resolved histone modification profiling on microfluidic platforms. First, I applied Microfluidic Oscillatory Washing-based Chromatin Immunoprecipitation followed by sequencing (MOWChIP-seq) to study the effect of culture dimensionality, hypoxia stress and bacterium infection on histone modification landscapes of brain tumor cells. I identified differentially marked regions between different culture conditions. Second, I adapted indexed ChIPmentation and introduced mu-CM, a low-input microfluidic device capable of performing 8 assays in parallel on different histone marks using as few as 20 cells in less than 7 hours. Last, I investigated the spatially resolved epigenome and transcriptome of neuronal and glial cells from coronal sections of adult mouse neocortex. I applied unsupervised clustering to identify distinct spatial patterns in neocortex epigenome and transcriptome that were associated with central nervous system development. I demonstrated that our method is well suited for scarce samples, such as biopsy samples from patients in the context of precision medicine.Doctor of Philosophy
Epigenetic is the study of alternations in organisms not caused by alternation of the genetic codes. Epigenetic information plays pivotal role during growth, aging and disease. Epigenetic information is dynamic and modifiable, and thus serves as an ideal target for various diagnostic and therapeutic strategies of human diseases. Microfluidics is a technology that manipulates liquids with extremely small volumes in miniaturized devices. Microfluidics has improved the sensitivity and resolution of epigenetic analysis. In this thesis, I report three projects focusing on low-input, cell-type-specific and spatially resolved histone modification profiling on microfluidic platforms. Histone modification is one type of epigenetic information and regulates gene expression. First, we studied the influence of culture condition and bacterium infection on histone modification profile of brain tumor cells. Second, we introduced mu-CM, combining a low-input microfluidic device with indexed ChIPmentation and is capable of performing 8 assays in parallel using as few as 20 cells. Last, we investigated spatial variations in the epigenome and transcriptome across adult mouse neocortex, the outer layer of brain involving in higher-order function, such as cognition. I identified distinct spatial patterns responsible for central nervous system development using machine learning algorithm. Our method is well suited for studying scarce samples, such as cells populations isolated from patients in the context of precision medicine.
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Hunt, Spencer Philip. « Whole-Genome Assembly of Atriplex hortensis L. Using OxfordNanopore Technology with Chromatin-Contact Mapping ». BYU ScholarsArchive, 2019. https://scholarsarchive.byu.edu/etd/8580.
Texte intégralRésumé :
Atriplex hortensis (2n = 2x = 18, 1C genome size ~1.1 gigabases), also known as garden orach, is a highly nutritious, broadleaf annual of the Amaranthaceae-Chenopodiaceae family that has spread from its native Eurasia to other temperate and subtropical environments worldwide. Atriplex is a highly complex and polyphyletic genus of generally halophytic and/or xerophytic plants, some of which have been used as food sources for humans and animals alike. Although there is some literature describing the taxonomy and ecology of orach, there is a lack of genetic and genomic data that would otherwise help elucidate the genetic variation, phylogenetic position, and future potential of this species. Here, we report the assembly of the first highquality, chromosome-scale reference genome for orach cv. ‘Golden’. Sequence data was produced using Oxford Nanopore’s MinION sequencing technology in conjunction with Illumina short-reads and chromatin-contact mapping. Genome assembly was accomplished using the high-noise, single-molecule sequencing assembler, Canu. The genome is enriched for highly repetitive DNA (68%). The Canu assembly combined with the Hi-C chromatin-proximity data yielded a final assembly containing 1,325 scaffolds with a contig N50 of 98.9 Mb and with 94.7% of the assembly represented in the nine largest, chromosome-scale scaffolds. Sixty-eight percent of the genome was classified as highly repetitive DNA, with the most common repetitive elements being Gypsy and Copia-like LTRs. The annotation was completed using MAKER which identified 31,010 gene models and 2,555 tRNA genes. Completeness of the genome was assessed using the Benchmarking Universal Single Copy Orthologs (BUSCO) platform, which quantifies functional gene content using a large core set of highly conserved orthologous genes (COGs). Of the 1,375 plant-specific COGs in the Embryophyta database, 1,330 (96.7%) were identified in the Atriplex assembly. We also report the results of a resequencing panel consisting of 21 accessions which illustrates a high degree of genetic similarity among cultivars and wild material from various locations in North America and Europe. These genome resources provide vital information to better understand orach and facilitate future study and comparison.
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Kremsky, Isaac Jacob 1983. « Assessing the relationship between chromatin and splicing factors in alternative splicing ». Doctoral thesis, Universitat Pompeu Fabra, 2015. http://hdl.handle.net/10803/316790.
Texte intégralRésumé :
Proteins that bind to DNA or RNA are both known to influence alternative splicing.
However, there has not been so far a systematic experimental exploration of the
relationship between these factors in their effect on splicing. In this thesis, we make use
of the large amounts of publicly available high throughput sequencing data that now
make it possible to explore this question on a genome-wide scale. We made exhaustive
use of a method known as profiling to address this question. As most profiling methods
in common use are merely qualitative, the first task of the thesis was to generate a
quantitative profiling method and bioinformatics tool, ProfileSeq, which we validated
by reproducing previous results from the literature. ProfileSeq and other methods were
combined to mine for relationships between DNA and RNA binding factors with
potential relevance to splicing. We found significant associations between the
transcription factor CTCF and the RNA binding protein LIN28A, and similarly between
SPI1 and RNA-binding proteins that bind to AC-rich motifs, such as hnRNPL. These
represent putative relationships relevant to splicing, as these results were reached by
more than one independent method with independent datasets. We also show evidence
that CTCF acts as a barrier between regions of H3K4me3 marking inside genes. A
number of other results of potential interest to both the bioinformatics and molecular
biology communities are also describedLas proteínas que se unen al DNA o al RNA pueden influir el splicing alternativo. Sin embargo, no ha habido aún una exploración sistemática de la relación entre estos dos tipos de factores en su acción sobre el splicing. En esta tesis hacemos uso de datos públicos de secuenciación de alto rendimiento para explorar esta cuestión a escala de todo el genoma. Hemos hecho un uso sistemático de la construcción de perfiles de información genómica para abordar esta cuestión. Debido a que los métodos i comúnmente utilizados para construir perfiles hace sólo comparaciones cualitativas, la primera tarea de esta tesis consistió en desarrollar un método para cuantificar perfiles e implementarlo en una herramienta bioinformática, ProfileSeq, la cual hemos validado mediante la reproducción de resultados previamente descritos en la literatura. Posteriormente, ProfileSeq se usó con datos de actividad de unión al DNA o al RNA de distintas proteínas para estudiar la relevancia en el splicing. Se encontraron varias asociaciones significativas. Entre ellas, la del factor de transcripción CTCF y la proteína de unión a RNA LIN28A. De manera similar, se encontró una relación entre SPI1 y proteínas de unión a RNA que se unen a motivos ricos en AC, como hnRNPL. Estos resultados representan relaciones putativas relevantes para el splicing, ya que se alcanzaron por más de un método diferente y usando datos independientes, También mostramos evidencia de que CTCF actúa como una barrera entre las regiones intragénicas de marcaje diferencial con H3K4me3. También se describen otros resultados de interés potencial tanto para la bioinformática como para la biología molecular.
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Sarma, Mimosa. « Microfluidic platforms for Transcriptomics and Epigenomics ». Diss., Virginia Tech, 2019. http://hdl.handle.net/10919/90294.
Texte intégralRésumé :
A cell, the building block of all life, stores a plethora of information in its genome, epigenome, and transcriptome which needs to be analyzed via various Omic studies. The heterogeneity in a seemingly similar group of cells is an important factor to consider and it could lead us to better understand processes such as cancer development and resistance to treatment, fetal development, and immune response. There is an ever growing demand to be able to develop more sensitive, accurate and robust ways to study Omic information and to analyze subtle biological variation between samples even with limited starting material obtained from a single cell. Microfluidics has opened up new and exciting possibilities that have revolutionized how we study and manipulate the contents of the cell like the DNA, RNA, proteins, etc. Microfluidics in conjunction with Next Gen Sequencing has provided ground-breaking capabilities for handling small sample volumes and has also provided scope for automation and multiplexing. In this thesis, we discuss a number of platforms for developing low-input or single cell Omic technologies. The first part talks about the development of a novel microfluidic platform to carry out single-cell RNA-sequencing in a one-pot method with a diffusion-based reagent swapping scheme. This platform helps to overcome the limitations of conventional microfluidic RNA seq methods reported in literature that use complicated multiple-chambered devices. It also provides good quality data that is comparable to state-of-the-art scRNA-seq methods while implementing a simpler device design that permits multiplexing. The second part talks about studying the transcriptome of innate leukocytes treated with varying levels of LPS and using RNA-seq to observe how innate immune cells undergo epigenetic reprogramming to develop phenotypes of memory cells. The third part discusses a low-cost alternative to produce tn5 enzyme which low-cost NGS studies. And finally, we discuss a microfluidic approach to carrying out low-input epigenomic studies for studying transcription factors. Today, single-cell or low-input Omic studies are rapidly moving into the clinical setting to enable studies of patient samples for personalized medicine. Our approaches and platforms will no doubt be important for transcriptomic and epigenomic studies of scarce cell samples under such settings.Doctor of Philosophy
This is the era of personalized medicine which means that we are no longer looking at one-size-fits-all therapies. We are rather focused on finding therapies that are tailormade to every individual’s personal needs. This has become more and more essential in the context of serious diseases like cancer where therapies have a lot of side-effects. To provide tailor-made therapy to patients, it is important to know how each patient is different from another. This difference can be found from studying how the individual is unique or different at the cellular level i.e. by looking into the contents of the cell like DNA, RNA, and chromatin. In this thesis, we discussed a number of projects which we can contribute to advancement in this field of personalized medicine. Our first project, MID-RNA-seq offers a new platform for studying the information contained in the RNA of a single cell. This platform has enough potential to be scaled up and automated into an excellent platform for studying the RNA of rare or limited patient samples. The second project discussed in this thesis involves studying the RNA of innate immune cells which defend our bodies against pathogens. The RNA data that we have unearthed in this project provides an immense scope for understanding innate immunity. This data provides our biologist collaborators the scope to test various pathways in innate immune cells and their roles in innate immune modulation. Our third project discusses a method to produce an enzyme called ‘Tn5’ which is necessary for studying the sequence of DNA. This enzyme which is commercially available has a very high cost associated with it but because we produced it in the lab, we were able to greatly reduce costs. The fourth project discussed involves the study of chromatin structure in cells and enables us to understand how our lifestyle choices change the expression or repression of genes in the cell, a study called epigenetics. The findings of this study would enable us to study epigenomic profiles from limited patient samples. Overall, our projects have enabled us to understand the information from cells especially when we have limited cell numbers. Once we have all this information we can compare how each patient is different from others. The future brings us closer to putting this into clinical practice and assigning different therapies to patients based on such data.
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Tavernari, Daniele. « Statistical and network-based methods for the analysis of chromatin accessibility maps in single cells ». Master's thesis, Alma Mater Studiorum - Università di Bologna, 2016. http://amslaurea.unibo.it/12297/.
Texte intégralRésumé :
In questo lavoro, metodi provenienti dalla Fisica, dalla Statistica e dalla Teoria dei Grafi sono stati impiegati per caratterizzare ed analizzare profili di apertura e accessibilità della cromatina ottenuti con la tecnica ATAC-seq in singole cellule, nella fattispecie linfociti B provenienti da tre pazienti affetti da Leucemia Linfocitica Cronica.
Una pipeline bioinformatica è stata sviluppata per processare i dati di sequencing ed ottenere le posizioni accessibili del genoma per ciascuna cellula. La quantità di regioni aperte e la loro distribuzione spaziale lungo il DNA sono state caratterizzate. Infine, l’apertura simultanea nelle stesse singole cellule di regioni regolatrici è stata impiegata come metrica per valutare relazioni funzionali, e in questo modo grafi tra enhancer e promoter sono stati costruiti e le loro proprietà sono state analizzate.
La distribuzione spaziale lungo il genoma di regioni aperte consecutive ricapitola proprietà strutturali come gli array di nucleosomi e le strutture a loop della cromatina. Inoltre, i profili di accessibilità delle regioni regolatrici sono significativamente conservati nelle singole cellule. I network tra enhancer e promoter forniscono un modo per caratterizzare la rilevanza di ciascuna regione regolatrice in termini di centralità. Le statistiche sulla connettività tra enhancer e promoter confermano il modello di relazione uno-a-uno come il più frequente, in cui un promoter è regolato dall'enhancer ad esso più vicino. Infine, anche il funzionamento dei superenhancer è stato indagato.
In conclusione, ATAC-seq si rivela un'efficace tecnica per indagare l'apertura della cromatina in singole cellule, i cui profili di accessibilità ricapitolano caratteristiche strutturali e funzionali della cromatina. Al fine di indagare i meccanismi della malattia, il panorama di accessibilità dei lifociti tumorali può essere confrontato con quello di cellule sane e cellule trattate con farmaci epigenetici.
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Ma, Sai. « Microfluidics for Genetic and Epigenetic Analysis ». Diss., Virginia Tech, 2017. http://hdl.handle.net/10919/78187.
Texte intégralRésumé :
Microfluidics has revolutionized how molecular biology studies are conducted. It permits profiling of genomic and epigenomic features for a wide range of applications. Microfluidics has been proven to be highly complementary to NGS technology with its unique capabilities for handling small volumes of samples and providing platforms for automation, integration, and multiplexing. In this thesis, we focus on three projects (diffusion-based PCR, MID-RRBS, and SurfaceChIP-seq), which improved the sensitivities of conventional assays by coupling with microfluidic technology. MID-RRBS and SurfaceChIP-seq projects were designed to profiling genome-wide DNA methylation and histone modifications, respectively. These assays dramatically improved the sensitivities of conventional approaches over 1000 times without compromising genomic coverages. We applied these assays to examine the neuronal/glial nuclei isolated from mouse brain tissues. We successfully identified the distinctive epigenomic signatures from neurons and glia. Another focus of this thesis is applying electrical field to investigate the intracellular contents. We report two projects, drug delivery to encapsulated bacteria and mRNA extraction under ultra-high electrical field intensity. We envision rapid growth in these directions, driven by the needs for testing scarce primary cells samples from patients in the context of precision medicine.Ph. D.
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Herzel, Lydia. « Co-transcriptional splicing in two yeasts ». Doctoral thesis, Saechsische Landesbibliothek- Staats- und Universitaetsbibliothek Dresden, 2015. http://nbn-resolving.de/urn:nbn:de:bsz:14-qucosa-179274.
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Cellular function and physiology are largely established through regulated gene expression. The first step in gene expression, transcription of the genomic DNA into RNA, is a process that is highly aligned at the levels of initiation, elongation and termination. In eukaryotes, protein-coding genes are exclusively transcribed by RNA polymerase II (Pol II). Upon transcription of the first 15-20 nucleotides (nt), the emerging nascent RNA 5’ end is modified with a 7-methylguanosyl cap. This is one of several RNA modifications and processing steps that take place during transcription, i.e. co-transcriptionally. For example, protein-coding sequences (exons) are often disrupted by non-coding sequences (introns) that are removed by RNA splicing. The two transesterification reactions required for RNA splicing are catalyzed through the action of a large macromolecular machine, the spliceosome. Several non-coding small nuclear RNAs (snRNAs) and proteins form functional spliceosomal subcomplexes, termed snRNPs.
Sequentially with intron synthesis different snRNPs recognize sequence elements within introns, first the 5’ splice site (5‘ SS) at the intron start, then the branchpoint and at the end the 3’ splice site (3‘ SS). Multiple conformational changes and concerted assembly steps lead to formation of the active spliceosome, cleavage of the exon-intron junction, intron lariat formation and finally exon-exon ligation with cleavage of the 3’ intron-exon junction. Estimates on pre-mRNA splicing duration range from 15 sec to several minutes or, in terms of distance relative to the 3‘ SS, the earliest detected splicing events were 500 nt downstream of the 3‘ SS. However, the use of indirect assays, model genes and transcription induction/blocking leave the question of when pre-mRNA splicing of endogenous transcripts occurs unanswered.
In recent years, global studies concluded that the majority of introns are removed during the course of transcription. In principal, co-transcriptional splicing reduces the need for post-transcriptional processing of the pre-mRNA. This could allow for quicker transcriptional responses to stimuli and optimal coordination between the different steps. In order to gain insight into how pre-mRNA splicing might be functionally linked to transcription, I wanted to determine when co-transcriptional splicing occurs, how transcripts with multiple introns are spliced and if and how the transcription termination process is influenced by pre-mRNA splicing.
I chose two yeast species, S. cerevisiae and S. pombe, to study co-transcriptional splicing. Small genomes, short genes and introns, but very different number of intron-containing genes and multi-intron genes in S. pombe, made the combination of both model organisms a promising system to study by next-generation sequencing and to learn about co-transcriptional splicing in a broad context with applicability to other species. I used nascent RNA-Seq to characterize co-transcriptional splicing in S. pombe and developed two strategies to obtain single-molecule information on co-transcriptional splicing of endogenous genes:
(1) with paired-end short read sequencing, I obtained the 3’ nascent transcript ends, which reflect the position of Pol II molecules during transcription, and the splicing status of the nascent RNAs. This is detected by sequencing the exon-intron or exon-exon junctions of the transcripts. Thus, this strategy links Pol II position with intron splicing of nascent RNA. The increase in the fraction of spliced transcripts with further distance from the intron end provides valuable information on when co-transcriptional splicing occurs.
(2) with Pacific Biosciences sequencing (PacBio) of full-length nascent RNA, it is possible to determine the splicing pattern of transcripts with multiple introns, e.g. sequentially with transcription or also non-sequentially. Part of transcription termination is cleavage of the nascent transcript at the polyA site. The splicing status of cleaved and non-cleaved transcripts can provide insights into links between splicing and transcription termination and can be obtained from PacBio data.
I found that co-transcriptional splicing in S. pombe is similarly prevalent to other species and that most introns are removed co-transcriptionally. Co-transcriptional splicing levels are dependent on intron position, adjacent exon length, and GC-content, but not splice site sequence. A high level of co-transcriptional splicing is correlated with high gene expression. In addition, I identified low abundance circular RNAs in intron-containing, as well as intronless genes, which could be side-products of RNA transcription and splicing.
The analysis of co-transcriptional splicing patterns of 88 endogenous S. cerevisiae genes showed that the majority of intron splicing occurs within 100 nt downstream of the 3‘ SS. Saturation levels vary, and confirm results of a previous study. The onset of splicing is very close to the transcribing polymerase (within 27 nt) and implies that spliceosome assembly and conformational rearrangements must be completed immediately upon synthesis of the 3‘ SS.
For S. pombe genes with multiple introns, most detected transcripts were completely spliced or completely unspliced. A smaller fraction showed partial splicing with the first intron being most often not spliced. Close to the polyA site, most transcripts were spliced, however uncleaved transcripts were often completely unspliced. This suggests a beneficial influence of pre-mRNA splicing for efficient transcript termination.
Overall, sequencing of nascent RNA with the two strategies developed in this work offers significant potential for the analysis of co-transcriptional splicing, transcription termination and also RNA polymerase pausing by profiling nascent 3’ ends. I could define the position of pre-mRNA splicing during the process of transcription and provide evidence for fast and efficient co-transcriptional splicing in S. cerevisiae and S. pombe, which is associated with highly expressed genes in both organisms. Differences in S. pombe co-transcriptional splicing could be linked to gene architecture features, like intron position, GC-content and exon length.
Livres sur le sujet "Chromatin sequencing"
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Liang, Xiaoshan. Studies of rainbow trout Ki-ras gene : Sequencing, aflatoxin B1 binding, and chromatin structure. 1993.
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Mifsud, Borbala, Kathi Zarnack et Anaïs F. Bardet. Practical Guide to ChIP-Seq Data Analysis. Taylor & Francis Group, 2018.
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Mifsud, Borbala, Kathi Zarnack et Anais Bardet. Practical Guide to Chip-Seq Data Analysis. Taylor & Francis Group, 2021.
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Mifsud, Borbala, Kathi Zarnack et Anaïs F. Bardet. Practical Guide to ChIP-Seq Data Analysis. Taylor & Francis Group, 2018.
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Mifsud, Borbala, et Anais Bardet. Practical Guide to Chip-Seq Data Analysis. Taylor & Francis Group, 2018.
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Practical Guide to ChIP-Seq Data Analysis. Taylor & Francis Group, 2018.
Trouver le texte intégralChapitres de livres sur le sujet "Chromatin sequencing"
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Ribarska, Teodora, et Gregor D. Gilfillan. « Native Chromatin Immunoprecipitation-Sequencing (ChIP-Seq) from Low Cell Numbers ». Dans Chromatin Immunoprecipitation, 157–66. New York, NY : Springer New York, 2017. http://dx.doi.org/10.1007/978-1-4939-7380-4_14.
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Hoeijmakers, Wieteke Anna Maria, et Richárd Bártfai. « Characterization of the Nucleosome Landscape by Micrococcal Nuclease-Sequencing (MNase-seq) ». Dans Chromatin Immunoprecipitation, 83–101. New York, NY : Springer New York, 2017. http://dx.doi.org/10.1007/978-1-4939-7380-4_8.
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Ludwig, Leif S., et Caleb A. Lareau. « Concomitant Sequencing of Accessible Chromatin and Mitochondrial Genomes in Single Cells Using mtscATAC-Seq ». Dans Chromatin Accessibility, 269–82. New York, NY : Springer US, 2023. http://dx.doi.org/10.1007/978-1-0716-2899-7_14.
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Stewart-Morgan, Kathleen R., et Anja Groth. « Profiling Chromatin Accessibility on Replicated DNA with repli-ATAC-Seq ». Dans Chromatin Accessibility, 71–84. New York, NY : Springer US, 2023. http://dx.doi.org/10.1007/978-1-0716-2899-7_6.
Texte intégralRésumé :
AbstractOpen or accessible chromatin typifies euchromatic regions and helps define cell type-specific transcription programs. DNA replication massively disorders chromatin composition and structure, and how accessible regions are affected by and recover from this disruption has been unclear. Here, we present repli-ATAC-seq, a protocol to profile accessible chromatin genome-wide on replicated DNA starting from 100,000 cells. In this method, replicated DNA is labeled with a short 5-ethynyl-2′-deoxyuridine (EdU) pulse in cultured cells and isolated from a population of tagmented fragments for amplification and next-generation sequencing. Repli-ATAC-seq provides high-resolution information on chromatin dynamics after DNA replication and reveals new insights into the interplay between DNA replication, transcription, and the chromatin landscape.
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Soares, Mário A. F., et Diogo S. Castro. « Chromatin Immunoprecipitation from Mouse Embryonic Tissue or Adherent Cells in Culture, Followed by Next-Generation Sequencing ». Dans Chromatin Immunoprecipitation, 53–63. New York, NY : Springer New York, 2017. http://dx.doi.org/10.1007/978-1-4939-7380-4_5.
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Diaz, Roxanne E., Aurore Sanchez, Véronique Anton Le Berre et Jean-Yves Bouet. « High-Resolution Chromatin Immunoprecipitation : ChIP-Sequencing ». Dans The Bacterial Nucleoid, 61–73. New York, NY : Springer New York, 2017. http://dx.doi.org/10.1007/978-1-4939-7098-8_6.
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Brahma, Sandipan, et Steven Henikoff. « CUT&RUN Profiling of the Budding Yeast Epigenome ». Dans Methods in Molecular Biology, 129–47. New York, NY : Springer US, 2022. http://dx.doi.org/10.1007/978-1-0716-2257-5_9.
Texte intégralRésumé :
AbstractMapping the epigenome is key to describe the relationship between chromatin landscapes and the control of DNA-based cellular processes such as transcription. Cleavage under targets and release using nuclease (CUT&RUN) is an in situ chromatin profiling strategy in which controlled cleavage by antibody-targeted Micrococcal Nuclease solubilizes specific protein-DNA complexes for paired-end DNA sequencing. When applied to budding yeast, CUT&RUN profiling yields precise genome-wide maps of histone modifications, histone variants, transcription factors, and ATP-dependent chromatin remodelers, while avoiding cross-linking and solubilization issues associated with the most commonly used chromatin profiling technique Chromatin Immunoprecipitation (ChIP). Furthermore, targeted chromatin complexes cleanly released by CUT&RUN can be used as input for a subsequent native immunoprecipitation step (CUT&RUN.ChIP) to simultaneously map two epitopes in single molecules genome-wide. The intrinsically low background and high resolution of CUT&RUN and CUT&RUN.ChIP allows for identification of transient genomic features such as dynamic nucleosome-remodeling intermediates. Starting from cells, one can perform CUT&RUN or CUT&RUN.ChIP and obtain purified DNA for sequencing library preparation in 2 days.
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Sridhar, Divya, et Aziz Aboobaker. « Monitoring Chromatin Regulation in Planarians Using Chromatin Immunoprecipitation Followed by Sequencing (ChIP-seq) ». Dans Methods in Molecular Biology, 529–47. New York, NY : Springer US, 2022. http://dx.doi.org/10.1007/978-1-0716-2172-1_28.
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AbstractPlanarians are an accessible model system to study animal regeneration and stem cells. Over the last two decades, new molecular techniques have provided us with powerful tools to understand whole-body regeneration and pluripotent adult stem cells specifically. We describe a method for performing Chromatin Immunoprecipitation followed by sequencing (ChIP-seq) on planarian cells that relies on FACS to isolate different cell populations followed by immunoprecipitation and library preparation for next-generation sequencing. Whole-genome profiling of histone modifications enables a greater understanding of epigenetic mechanisms in development, pluripotency, and differentiation. This protocol adds to the growing list of functional genomic approaches to study whole-body regeneration in animals.
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Lopez-Rubio, Jose-Juan, T. Nicolai Siegel et Artur Scherf. « Genome-wide Chromatin Immunoprecipitation-Sequencing in Plasmodium ». Dans Methods in Molecular Biology, 321–33. Totowa, NJ : Humana Press, 2012. http://dx.doi.org/10.1007/978-1-62703-026-7_23.
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Ruan, Xiaoan, et Yijun Ruan. « Chromatin Interaction Analysis Using Paired-End Tag Sequencing (ChIA-PET) ». Dans Tag-Based Next Generation Sequencing, 185–210. Weinheim, Germany : Wiley-VCH Verlag GmbH & Co. KGaA, 2012. http://dx.doi.org/10.1002/9783527644582.ch12.
Texte intégralActes de conférences sur le sujet "Chromatin sequencing"
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Qiao, Yi, Xiaomeng Huang et Gabor Marth. « Abstract 40 : scBayes : A computational method to study tumor subclone-specific gene expression and chromatin accessibility using single-cell RNA sequencing and single-cell ATAC sequencing in combination of bulk DNA sequencing ». Dans Abstracts : AACR Special Conference on Advancing Precision Medicine Drug Development : Incorporation of Real-World Data and Other Novel Strategies ; January 9-12, 2020 ; San Diego, CA. American Association for Cancer Research, 2020. http://dx.doi.org/10.1158/1557-3265.advprecmed20-40.
Texte intégralRapports d'organisations sur le sujet "Chromatin sequencing"
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Gur, Amit, Edward Buckler, Joseph Burger, Yaakov Tadmor et Iftach Klapp. Characterization of genetic variation and yield heterosis in Cucumis melo. United States Department of Agriculture, janvier 2016. http://dx.doi.org/10.32747/2016.7600047.bard.
Texte intégralRésumé :
Project objectives: 1) Characterization of variation for yield heterosis in melon using Half-Diallele (HDA) design. 2) Development and implementation of image-based yield phenotyping in melon. 3) Characterization of genetic, epigenetic and transcriptional variation across 25 founder lines and selected hybrids. The epigentic part of this objective was modified during the course of the project: instead of characterization of chromatin structure in a single melon line through genome-wide mapping of nucleosomes using MNase-seq approach, we took advantage of rapid advancements in single-molecule sequencing and shifted the focus to Nanoporelong-read sequencing of all 25 founder lines. This analysis provides invaluable information on genome-wide structural variation across our diversity 4) Integrated analyses and development of prediction models Agricultural heterosis relates to hybrids that outperform their inbred parents for yield. First generation (F1) hybrids are produced in many crop species and it is estimated that heterosis increases yield by 15-30% globally. Melon (Cucumismelo) is an economically important species of The Cucurbitaceae family and is among the most important fleshy fruits for fresh consumption Worldwide. The major goal of this project was to explore the patterns and magnitude of yield heterosis in melon and link it to whole genome sequence variation. A core subset of 25 diverse lines was selected from the Newe-Yaar melon diversity panel for whole-genome re-sequencing (WGS) and test-crosses, to produce structured half-diallele design of 300 F1 hybrids (MelHDA25). Yield variation was measured in replicated yield trials at the whole-plant and at the rootstock levels (through a common-scion grafted experiments), across the F1s and parental lines. As part of this project we also developed an algorithmic pipeline for detection and yield estimation of melons from aerial-images, towards future implementation of such high throughput, cost-effective method for remote yield evaluation in open-field melons. We found extensive, highly heritable root-derived yield variation across the diallele population that was characterized by prominent best-parent heterosis (BPH), where hybrids rootstocks outperformed their parents by 38% and 56 % under optimal irrigation and drought- stress, respectively. Through integration of the genotypic data (~4,000,000 SNPs) and yield analyses we show that root-derived hybrids yield is independent of parental genetic distance. However, we mapped novel root-derived yield QTLs through genome-wide association (GWA) analysis and a multi-QTLs model explained more than 45% of the hybrids yield variation, providing a potential route for marker-assisted hybrid rootstock breeding. Four selected hybrid rootstocks are further studied under multiple scion varieties and their validated positive effect on yield performance is now leading to ongoing evaluation of their commercial potential. On the genomic level, this project resulted in 3 layers of data: 1) whole-genome short-read Illumina sequencing (30X) of the 25 founder lines provided us with 25 genome alignments and high-density melon HapMap that is already shown to be an effective resource for QTL annotation and candidate gene analysis in melon. 2) fast advancements in long-read single-molecule sequencing allowed us to shift focus towards this technology and generate ~50X Nanoporesequencing of the 25 founders which in combination with the short-read data now enable de novo assembly of the 25 genomes that will soon lead to construction of the first melon pan-genome. 3) Transcriptomic (3' RNA-Seq) analysis of several selected hybrids and their parents provide preliminary information on differentially expressed genes that can be further used to explain the root-derived yield variation. Taken together, this project expanded our view on yield heterosis in melon with novel specific insights on root-derived yield heterosis. To our knowledge, thus far this is the largest systematic genetic analysis of rootstock effects on yield heterosis in cucurbits or any other crop plant, and our results are now translated into potential breeding applications. The genomic resources that were developed as part of this project are putting melon in the forefront of genomic research and will continue to be useful tool for the cucurbits community in years to come.