Biology subjects

Garber, M.

Publications and source records attributed to Garber, M..

At least 19 recordsLinked to original sources

bioRxiv2025

Neutrophil Transcriptomics in SLE: Exploring Intrinsic, Ex Vivo Adaptation, and CAR T-Cell Therapy-Induced Changes

ObjectivesSystemic lupus erythematosus (SLE) is an autoimmune disease characterized by dysregulation of the adaptive and innate immunityThis study aimed to identify transcriptomic differences in neutrophils from SLE patients and healthy individuals, analyze ex vivo adaptation dynamics, and evaluate the impact of chimeric antigen receptor (CAR) T-cell therapy on neutrophil transcriptomic profiles. MethodsNeutrophils were isolated via negative selection from seven SLE patients and three healthy individuals. RNA sequencing was performed to assess transcriptomic differences, ex vivo dynamics over 60 minutes, and responses to lipopolysaccharide (LPS) stimulation. Additionally, longitudinal transcriptomic data from an SLE patient undergoing KYV-101 anti-CD19 CAR T-cell therapy were evaluated. ResultsWe identified 258 differentially expressed genes (DEGs) consistently distinguishing SLE from healthy neutrophils; they spanned multiple clusters, enriched in interferon-related and DNA damage repair genes (upregulated), and ribosomal protein genes (downregulated). Ex vivo adaptation revealed shared activation pathways, such as NF-{kappa}B and apoptosis, in both groups. LPS stimulation highlighted overlapping inflammatory responses, demonstrating retained functional capacities in SLE neutrophils. Following CAR T-cell therapy of an SLE patient,neutrophil transcriptomic profiles realigned with healthy controls by three months post-treatment. ConclusionsNeutrophils in SLE exhibit intrinsic, disease-specific transcriptomic alterations while sharing ex vivo adaptation dynamics with healthy individuals. The disease-specific alterations appear to be modifiable through targeted therapeutic intervention, as anti-CD19 CAR T-cell therapy resets neutrophil gene expression toward healthy patterns despite targeting B cells rather than neutrophils directly. These findings provide insights into SLE pathogenesis and highlight potential therapeutic strategies targeting both adaptive and innate immunity.

immunology↗

bioRxiv2025

Human Cytomegalovirus Infection of Primary Human Oral Keratinocytes Induces Intermediate Keratinocyte Differentiation and an Altered Innate Immune Response

The oral mucosa is central for human cytomegalovirus (HCMV) acquisition. However, the initial infection events in the oral cavity, including how HCMV modulates oral cells to establish infection and spread, are supported by limited experimental data and therefore remain poorly understood. We investigated HCMV infection in cultures of primary human oral keratinocytes (HOKs) as a model to study HCMV infection in the oral mucosa using the TB40E-Gfp and MOLD strains of HCMV. Both viral strains successfully infected and replicated in HOKs as demonstrated by the appearance of cytopathic effects, full expression of viral transcripts, and the shedding of infectious virus. To investigate how HCMV modulates the transcriptome of primary HOKs, we performed single cell RNA sequencing (scRNA-seq) which revealed 11 subtypes of cells. Furthermore, infection resulted in significant changes in host gene expression. HCMV upregulated genes involved in immune responses, cell cycle regulation, cancer-related pathways, neuronal and synaptic functions, metabolism, stress responses, molecular chaperones, and vesicular trafficking which might be critical for viral protein expression and assembly. HCMV also increased the expression of genes related to the extracellular matrix, cell adhesion, microtubules, signal transduction, kinases, and transcriptional regulation. Conversely, HCMV downregulated genes associated with inflammation and immune responses, cell cycle control and apoptosis, cell adhesion and migration, as well as signaling pathways, growth factors, ion channels, transporters, and transcription factors when compared to uninfected cells. These findings suggest that HCMV modulates a wide range of host cellular pathways to create a favorable environment for its replication. HCMV also induced changes in HOK differentiation genes downregulating basal state genes and upregulating intermediate genes and select terminal differentiation genes, indicating HCMV might be driving HOK differentiation towards a dysregulated intermediate phenotype while inhibiting their terminal differentiation to maintain a cell state capable of sustaining viral replication. Using SciViewer, our recently developed scRNA-seq data visualization tool, we determined that as the infection progressed from low to high viral transcript accumulation, HCMV infection upregulated E2F targeted genes, some antiviral and immune responses as well as autophagy and cellular stress responses, while downregulating interferon-stimulated genes, immune and antiviral response genes, pyroptosis, inflammatory cell death, and membrane remodeling and viral entry genes. Overall, our study shows that primary human HOKs can be used as a model to study HCMV infection in the oral mucosa and that HCMV drives dysregulated intermediate oral keratinocyte differentiation and altered immune responses which likely facilitate viral replication in the oral mucosa and spread into the rest of the host.

molecular biology↗

bioRxiv2025

A Spatially Coordinated Keratinocyte-Fibroblast Circuit Recruits MMP9+ Myeloid Cells to Drive IFN-I-Driven Inflammation in Photosensitive Autoimmunity

Photosensitivity is a hallmark of cutaneous lupus erythematosus (CLE) and dermatomyositis (DM), yet the mechanisms linking ultraviolet B (UVB) exposure to tissue-specific autoimmunity remain incompletely defined. Here, we use an integrative human-based approach, including single-cell RNA sequencing, spatial transcriptomics (seqFISH+), in vivo UVB provocation, and in vitro modeling, to uncover a spatially coordinated inflammatory circuit that underlies interferon-I (IFN-I)-amplified skin pathology. We identify MMP9+ CD14+ myeloid cells as central effectors of photosensitivity in both CLE and DM. These cells are markedly expanded in lesional skin, serve as the dominant source of IFN-{beta}, and colocalize with cytotoxic CD4+ T cells at the dermal-epidermal junction. Spatial transcriptomics further reveals a keratinocyte-fibroblast-myeloid axis, wherein keratinocytes activate discrete subsets of pro-inflammatory fibroblasts in the superficial dermis to produce monocyte-attracting chemokines, including CCL2, CCL19, CCL7, CCL8, and CXCL12, directing MMP9+ CD14+ cell recruitment toward the interface. In our in-vitro model, IFN-I-primed basal keratinocytes undergo heightened UVB-induced cell death and release membrane-associated cytokines such as TNF-, IL-1, which activate monocyte-derived dendritic cells (moDCs) and induce transcriptional programs mirroring those of MMP9+ CD14+ cells in vivo. In vivo, UVB irradiation of non-lesional DM skin, but not healthy controls, elicits rapid infiltration of these myeloid cells, confirming their disease-specific responsiveness to UVB. Finally, in a proof-of-concept clinical study, treatment with anifrolumab (anti-IFN-I receptor) blocked UVB-induced MMP9+ CD14+ infiltration and attenuated photosensitivity in CLE. Together, these findings define a multicellular inflammatory cascade linking keratinocyte injury, fibroblast chemotactic programming, and myeloid effector function in IFN-I-driven skin autoimmunity and nominate MMP9+ CD14+ cells as actionable targets in photosensitive dermatoses. Photosensitivity is central to cutaneous lupus erythematosus (CLE) and dermatomyositis (DM), but the mechanisms linking UVB exposure to tissue-specific autoimmunity are poorly defined. Using single-cell RNA sequencing, spatial transcriptomics, UVB provocation, and in vitro modeling, we identify MMP9+ CD14+ myeloid cells as critical mediators of photosensitivity. These cells expand significantly in lesional skin, produce IFN-{beta}, and colocalize with cytotoxic CD4+ T cells at the dermal-epidermal junction. Keratinocytes activate fibroblasts in the superficial dermis, prompting them to release chemokines (CCL2, CCL19, CCL7, CCL8, CXCL12) that recruit MMP9+ CD14+ cells. IFN-I-primed keratinocytes exposed to UVB release cytokines activating dendritic cells, mirroring in vivo responses. UVB irradiation of non-lesional DM skin rapidly recruits these myeloid cells. In a clinical proof-of-concept study, anti-IFN-I treatment with anifrolumab prevented UVB-induced myeloid infiltration and reduced photosensitivity. Thus, targeting MMP9+ CD14+ cells may offer therapeutic potential for managing photosensitive autoimmune skin conditions.

immunology↗

bioRxiv2025

Endothelial Cell Expression of STINGV154M Gain-of-Function Mutation Delays the Resolution of UVB-induced Skin Injury

Gain-of-function mutations in STimulator of INterferon Genes (STING) cause STING-Associated Vasculopathy with Onset in Infancy (SAVI), a rare autoinflammatory disease characterized by debilitating inflammatory lung disease and hallmark skin manifestations, such as chilblains and progressive, non-healing ulcers. Mice expressing the most common SAVI-associated variant STINGV154M (VM) recapitulate many clinical features of SAVI, including inflammatory lung disease, but do not develop spontaneous skin lesions. In this study, we show that a single low dose of ultraviolet B (UVB) irradiation, which induces only transient skin inflammation in wild-type (WT) mice, causes severe and progressive skin injury in VM mice. Notably, this phenotype persisted in VM mice depleted of hematopoietic cells and reconstituted with WT bone marrow, demonstrating that STINGV154M expression in non-hematopoietic cells is sufficient to drive persistent skin inflammation. Further analysis identified endothelial cells expressing STINGV154M as the primary driver of the cutaneous phenotype. Flow cytometry and bulk RNA sequencing showed that VM mice exhibited reduced early skin infiltration of macrophages and dendritic cells after UVB exposure. These findings establish a critical link between endothelial STING activation, impaired recruitment of skin myeloid cells, and defective resolution of acute inflammation, offering new insights into the pathogenesis of SAVI-associated skin disease.

immunology↗

bioRxiv2025

Scalable TCR synthesis and screening enables antigen reactivity mapping in vitiligo

T cell receptors (TCRs) mediate antigen recognition in adaptive immunity, yet large-scale mapping of TCR-antigen interactions remains a major challenge. Current approaches to synthesize and functionally screen TCRs remain technically complex and limited in throughput. We introduce a modular strategy, TCRAFT, to rapidly construct tens of thousands of TCRs for <$1 each while maintaining TCR-{beta} pairing with >99% accuracy. We integrate this approach with a high-throughput antigen discovery platform to enable library-on-library TCR-antigen screening. We reconstruct and screen 3,808 TCRs from vitiligo lesions, linking TCR specificity to transcriptional phenotypes for antigen-reactive T cells. To demonstrate scalability, we synthesize and screen 30,810 TCRs from donors with pancreatic ductal adenocarcinoma to capture antigen-specific TCRs. This workflow reduces the cost and complexity of large-scale TCR screening, enabling the expansion of the known landscape of antigen-specific TCRs in vitiligo with a method that can be readily extended to other immunological applications.

immunology↗

bioRxiv2025

Worm Perturb-Seq: massively parallel whole-animal RNAi and RNA-seq

The transcriptome provides a highly informative molecular phenotype to connect genotype to phenotype and is most frequently measured by RNA-sequencing (RNA-seq). Therefore, an ultimate goal is to perturb every gene and measure changes in the transcriptome. However, this remains challenging, especially in intact organisms due to different experimental and computational challenges. Here, we present Worm Perturb-Seq (WPS), which provides high-resolution RNA-seq profiles for hundreds of replicate perturbations at a time in a living animal. WPS introduces multiple experimental advances that combine strengths of bulk and single cell RNA-seq, and that further provides an analytical framework, EmpirDE, that leverages the unique power of the large WPS datasets. EmpirDE identifies differentially expressed genes (DEGs) by using gene-specific empirical null distributions, rather than control conditions alone, thereby systematically removing technical biases and improving statistical rigor. We applied WPS to 103 Caenhorhabditis elegans nuclear hormone receptors (NHRs) to delineate a Gene Regulatory Network (GRN) and found that this GRN presents a striking pairwise modularity where pairs of NHRs regulate shared target genes. We envision that the experimental and analytical advances of WPS should be useful not only for C. elegans, but will be broadly applicable to other models, including human cells.

genomics↗

bioRxiv2024

A single cell atlas of the mouse seminal vesicle

During mammalian reproduction, sperm are delivered to the female reproductive tract bathed in a complex medium known as seminal fluid, which plays key roles in signaling to the female reproductive tract and in nourishing sperm for their onwards journey. Along with minor contributions from the prostate and the epididymis, the majority of seminal fluid is produced by a somewhat understudied organ known as the seminal vesicle. Here, we report the first single-cell RNA-seq atlas of the mouse seminal vesicle, generated using tissues obtained from 23 mice of varying ages, exposed to a range of dietary challenges. We define the transcriptome of the secretory cells in this tissue, identifying a relatively homogeneous population of the epithelial cells which are responsible for producing the majority of seminal fluid. We also define the immune cell populations - including large populations of macrophages, dendritic cells, T cells, and NKT cells - which have the potential to play roles in producing various immune mediators present in seminal plasma. Together, our data provide a resource for understanding the composition of an understudied reproductive tissue with potential implications for paternal control of offspring development and metabolism.

genomics↗

bioRxiv2024

Development of a high-throughput, quantitative platform using human cerebral organoids to study virus-induced neuroinflammation in Alzheimer's disease

Neuroinflammation is a central process in the pathogenesis of several neurodegenerative diseases such as Alzheimers disease (AD), and there are active efforts to target pathways involved in neuroinflammation for molecular biomarker discovery and therapeutic development in neurodegenerative diseases. It was also proposed that there may be an infectious etiology in AD that is associated with viruses such as herpes simplex virus (HSV-1) and influenza A virus (IAV), leading to neuroinflammation-induced AD pathogenesis or disease progression. We sought to develop high-throughput, quantitative molecular biomarker assays using dissociated cells from human cerebral organoids (dcOrgs), that can used for screening compounds to reverse AD-associated neuroinflammation. We found that HSV-1 infection, but not IAV infection, in dcOrgs led to increased intracellular A{beta}42 and phosphorylated Tau-Thr212 (pTau-212) expression, lower ratios of secreted A{beta}42/40, as well as neuronal loss, and increased proportions of astrocytes and microglia, which are hallmarks of AD. Among the glia cell-type markers, Iba1 (microglia) and GFAP (astrocyte) expression were most strongly correlated with HSV-1 expression, which further supported that these biomarkers are perturbed by glia-mediated neuroinflammation. By performing large-scale RNA sequencing, we observed that differentially expressed transcripts in HSV-1 infected dcOrgs were specifically enriched for AD-associated GWAS genes, but not for genes associated with other common neurodegenerative, neuropsychiatric or autoimmune diseases. Immediate treatment of HSV-1 infected dcOrgs with anti-herpetic drug acyclovir (ACV) rescued most of the cellular and transcriptomic biomarkers in a dosage-dependent manner, indicating that it is possible to use our high-throughput platform to identify compounds or target genes that can reverse these neuroinflammation-induced biomarkers associated with AD.

genomics↗

bioRxiv2021

The Unique Cytoarchitecture and Wiring of the Human Default Mode Network

The default mode network (DMN) is implicated in many aspects of complex thought and behavior. Here, we leverage post-mortem histology and in vivo neuroimaging to characterise the anatomy of the DMN to better understand its role in information processing and cortical communication. Our results show that the DMN is cytoarchitecturally heterogenous, containing cytoarchitectural types that are variably specialised for unimodal, heteromodal, and memory-related processing. Studying diffusion-based structural connectivity in combination with cytoarchitecture, we found the DMN contains regions receptive to input from sensory cortex and a core that is relatively insulated from environmental input. Finally, analysis of signal flow with effective connectivity models showed that the DMN is unique amongst cortical networks in balancing its output across the levels of sensory hierarchies. Together, our study establishes an anatomical foundation from which mechanistic accounts of the broad role the DMN plays in human brain function and cognition can be developed.

neuroscience↗

bioRxiv2021

Scalable and Automated CRISPR-Based Strain Engineering Using Droplet Microfluidics

We present a droplet-based microfluidic system that enables CRISPR-based gene editing and high-throughput screening on chip. The microfluidic device contains a 10 x 10 element array, each element containing sets of electrodes for two electric field actuated operations-electrowetting for merging droplets to mix reagents and electroporation for transformation. It can perform up to 100 genetic modifications in parallel, providing a scalable platform for generating the large number of engineered strains required for combinatorial optimization of genetic pathways and predictable bioengineering. We demonstrate the systems capabilities through CRISPR-based engineering of two test cases-1) disruption of the function of enzyme galactokinase (galK) in E. coli and 2) targeted engineering of glutamine synthetase gene (glnA) and blue-pigment synthetase (bpsA) enzyme to improve indigoidine production in E. coli.

synthetic biology↗

bioRxiv2021

Simultaneous profiling of multiple chromatin proteins in the same cells

Methods derived from CUT&RUN and CUT&Tag enable genome-wide mapping of the localization of proteins on chromatin from as few as one cell. These and other mapping approaches focus on one protein at a time, preventing direct measurements of colocalization of different chromatin proteins in the same cells and requiring prioritization of targets where samples are limiting. Here we describe multi-CUT&Tag, an adaptation of CUT&Tag that overcomes these hurdles by using antibody-specific barcodes to simultaneously map multiple proteins in the same cells. Highly specific multi-CUT&Tag maps of histone marks and RNA Polymerase II uncovered sites of co-localization in the same cells, active and repressed genes, and candidate cis-regulatory elements. Single-cell multi-CUT&Tag profiling facilitated identification of distinct cell types from a mixed population and characterization of cell type-specific chromatin architecture. In sum, multi-CUT&Tag increases the information content per cell of epigenomic maps, facilitating direct analysis of the interplay of different proteins on chromatin.

genomics↗

bioRxiv2020

The Tec kinase ITK differentially optimizes NFAT, NF-κB, and MAPK signaling during early T cell activation to regulate graded gene induction

The strength of peptide:MHC interactions with the T cell receptor (TCR) is correlated with the time to first cell division, the relative scale of the effector cell response, and the graded expression of activation-associated proteins like IRF4. To regulate T cell activation programming, the TCR and the TCR proximal kinase ITK simultaneously trigger many biochemically separate TCR signaling cascades. T cells lacking ITK exhibit selective impairments in effector T cell responses after activation, but under the strongest signaling conditions ITK activity is dispensable. To gain insight into whether TCR signal strength and ITK activity tune observed graded gene expression through unequal activation of disparate signaling pathways, we examined Erk1/2 activation and NFAT, NF-{kappa}B translocation in naive OT-I CD8+ cell nuclei. We observed consistent digital activation of NFAT1 and Erk-MAPK, but NF-{kappa}B displayed dynamic, graded activation in response to variation in TCR signal strength and was tunable by treatment with an ITK inhibitor. Inhibitor-treated cells showed dampened induction of AP-1 factors Fos and Fosb, NF-{kappa}B response gene transcripts, and survival factor Il2 transcripts. ATAC-seq analysis also revealed genomic regions most sensitive to ITK inhibition were enriched for NF-{kappa}B and AP-1 motifs. Specific inhibition of NF-{kappa}B during peptide stimulation tuned expression of early gene products like c-Fos. Together, these data indicate a key role for ITK in orchestrating optimal activation of separate TCR downstream pathways, specifically aiding NF-{kappa}B activation. More broadly, we revealed a mechanism by which variation in TCR signal strength can produce patterns of graded gene expression in activated T cells.

immunology↗

bioRxiv2020

Single-cell analysis of upper airway cells reveals host-viral dynamics in influenza infected adults

Influenza virus infections are major causes of morbidity and mortality. Research using cultured cells, bulk tissue, and animal models cannot fully capture human disease dynamics. Many aspects of virus-host interactions in a natural setting remain unclear, including the specific cell types that are infected and how they and neighboring bystander cells contribute to the overall antiviral response. To address these questions, we performed single-cell RNA sequencing (scRNA-Seq) on cells from freshly collected nasal washes from healthy human donors and donors diagnosed with acute influenza during the 2017-18 season. We describe a previously uncharacterized goblet cell population, specific to infected individuals, with high expression of MHC class II genes. Furthermore, leveraging scRNA-Seq reads, we obtained deep viral genome coverage and developed a model to rigorously identify infected cells that detected influenza infection in all epithelial cell types and even some immune cells. Our data revealed that each donor was infected by a unique influenza variant and that each variant was separated by at least one unique non-synonymous difference. Our results demonstrate the power of massively-parallel scRNA-Seq to study viral variation, as well as host and viral transcriptional activity during human infection.

genomics↗

bioRxiv2020

An atlas of cell types in the mammalian epididymis and vas deferens

Following spermatogenesis in the testis, mammalian sperm continue to mature over the course of approximately 10 days as they transit a long epithelial tube known as the epididymis. The epididymis is comprised of multiple segments/compartments that, in addition to concentrating sperm and preventing their premature activation, play key roles in remodeling the protein, lipid, and RNA composition of maturing sperm. In order to understand the complex roles for the epididymis in reproductive biology, we generated a single cell atlas of gene expression from the murine epididymis and vas deferens. We recovered all the key cell types of the epididymal epithelium, including principal cells, clear cells, and basal cells, along with associated support cells that include fibroblasts, smooth muscle, macrophages and other immune cells. Moreover, our data illuminate extensive regional specialization of principal cell populations across the length of the epididymis, with a substantial fraction of segment-specific genes localized in genomic clusters of functionally-related genes. In addition to the extensive region-specific specialization of principal cells, we find evidence for functionally-specialized subpopulations of stromal cells, and, most notably, two distinct populations of clear cells. Analysis of ligand/receptor expression reveals a network of potential cellular signaling connections, with several predicted interactions between cell types that may play roles in immune cell recruitment and other aspects of epididymal function. Our dataset extends on existing knowledge of epididymal biology, and provides a wealth of information on potential regulatory and signaling factors that bear future investigation.

developmental biology↗

bioRxiv2019

DolphinNext: A distributed data processing platform for high throughput genomics

The emergence of high throughput technologies that produce vast amounts of genomic data, such as next-generation sequencing (NGS) are transforming biological research. The dramatic increase in the volume of data makes analysis the main bottleneck for scientific discovery. The processing of high throughput datasets typically involves many different computational programs, each of which performs a specific step in a pipeline. Given the wide range of applications and organizational infrastructures, there is a great need for a highly-parallel, flexible, portable, and reproducible data processing frameworks. Flexibility ensures that pipelines can support a variety of applications without requiring one-off modifications. Portability ensures that users can leverage computationally available resources and work within economic constraints. Reproducibility warrants credibility to the results and is particularly challenging in the face of the sheer volume of data and the complexity of processing pipelines that vary widely between users.\n\nSeveral platforms currently exist for the design and execution of complex pipelines (e.g. Galaxy, GenePattern, GeneProf). Unfortunately, these platforms lack the necessary combination of parallelism, portability, flexibility and/or reproducibility that are required by the current research environment. To address these shortcomings, Nextflow was implemented to simplify portable, scalable, and reproducible scientific pipelines using containers. We have used Nextflow capabilities as leverage and developed a user interface, DolphinNext, for creating, deploying, and executing complex Nextflow pipelines for high throughput data processing. The guiding principle of DolphinNext is to facilitate the building and deployment of complex pipelines using a modular approach implemented in a graphical interface. DolphinNext provides: 1. A drag and drop user interface that abstracts pipelines and allows users to create pipelines without familiarity in underlying programming languages. 2. A user interface to monitor pipeline execution that allows the re-initiation of pipelines at intermediate steps 3. Reproducible pipelines with version tracking and stand-alone versions that can be run independently. 4. Seamless portability to distributed computational environments such as high-performance clusters or cloud computing environments.

bioinformatics↗

bioRxiv2019

Multiple human adipocyte subtypes and mechanisms of their development

Human adipose tissue depots perform numerous diverse physiological functions, and are differentially linked to metabolic disease risk, yet only two major human adipocyte subtypes have been described, white and "brown/brite/beige." The diversity and lineages of adipocyte classes have been studied in mice using genetic methods that cannot be applied in humans. Here we circumvent this problem by studying the fate of single mesenchymal progenitor cells obtained from human adipose tissue. We report that a minimum of four human adipocyte subtypes can be distinguished by transcriptomic analysis, specialized for functionally distinct processes such as adipokine secretion and thermogenesis. Evidence for the presence of these adipocytes subtypes in adult humans is evidenced by differential expression of key adipokines leptin and adiponectin in isolated mature adipocytes. The human adipocytes most similar to the mouse "brite/beige" adipocytes are enriched in mechanisms that promote iron accumulation and protect from oxidative stress, and are derived from progenitors that express high levels of cytokines such as IL1B, IL8, IL11 and the IL6 family cytokine LIF, and low levels of the transcriptional repressors ID1 and ID3. Our finding of this adipocyte repertoire and its developmental mechanisms provides a high-resolution framework to analyze human adipose tissue architecture and its role in systemic metabolism and metabolic disease.

physiology↗

bioRxiv2018

DEBrowser: Interactive Differential Expression Analysis and Visualization Tool for Count Data

BackgroundSequencing data has become a standard measure for studying diverse cellular activities. For example, gene expression is accurately measured by RNA sequencing (RNA-Seq) libraries, protein-DNA interactions are captured by chromatin immunoprecipitation sequencing (ChIP-Seq), protein-RNA interactions by crosslinking immunoprecipitation (CLIP-Seq) or RNA immunoprecipitation (RIP-Seq) sequencing, DNA accessibility by assay for transposase-accessible chromatin (ATAC-Seq), and DNase or MNase sequencing libraries. Analysis of these sequencing techniques involve library-specific approaches. However, in all cases, once the sequencing libraries are processed, the result is a count table specifying the estimated number of reads originating from a genomic locus. Differential analysis to determine which loci have different cellular activity under different conditions starts with the count table and iterates through a cycle of data assessment, preparation and analysis. Such iterative approach relies on multiple programs and is therefore a challenge for those without programming skills.\n\nResultsWe developed DEBrowser, as an R bioconductor project, to interactively visualize each step of the differential analysis of count data, without any requirement for programming expertise. The application presents a rich and interactive web based graphical user interface based on Rs shiny infrastructure. We use shinys reactive programming interface for a dynamic webpage that responds to user input and integrates its visualization widgets at each stage of the analysis. In this way, every step of the analysis can be displayed in one application that combines many approaches and multiple results. We show DEBrowsers capabilities by reproducing the analysis of two previously published data sets.\n\nConclusionsDEBrowser is a flexible, intuitive, web-based analysis platform that enables an iterative and interactive analysis of count data without any requirement of programming knowledge.

bioinformatics↗

bioRxiv2017

Higher-order inter-chromosomal hubs shape 3-dimensional genome organization in the nucleus

Eukaryotic genomes are packaged into a 3-dimensional structure in the nucleus of each cell. There are currently two distinct views of genome organization that are derived from different technologies. The first view, derived from genome-wide proximity ligation methods (e.g. Hi-C), suggests that genome organization is largely organized around chromosomes. The second view, derived from in situ imaging, suggests a central role for nuclear bodies. Yet, because microscopy and proximity-ligation methods measure different aspects of genome organization, these two views remain poorly reconciled and our overall understanding of how genomic DNA is organized within the nucleus remains incomplete. Here, we develop Split-Pool Recognition of Interactions by Tag Extension (SPRITE), which moves away from proximity-ligation and enables genome-wide detection of higher-order DNA interactions within the nucleus. Using SPRITE, we recapitulate known genome structures identified by Hi-C and show that the contact frequencies measured by SPRITE strongly correlate with the 3-dimensional distances measured by microscopy. In addition to known structures, SPRITE identifies two major hubs of inter-chromosomal interactions that are spatially arranged around the nucleolus and nuclear speckles, respectively. We find that the majority of genomic regions exhibit preferential spatial association relative to one of these nuclear bodies, with regions that are highly transcribed by RNA Polymerase II organizing around nuclear speckles and transcriptionally inactive and centromere-proximal regions organizing around the nucleolus. Together, our results reconcile the two distinct pictures of nuclear structure and demonstrate that nuclear bodies act as inter-chromosomal hubs that shape the overall 3-dimensional packaging of genomic DNA in the nucleus.

genomics↗