ORCID Profile
0000-0003-0772-1647
Current Organisation
New York Genome Center
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Publisher: Cold Spring Harbor Laboratory
Date: 28-08-2021
DOI: 10.1101/2021.08.27.457864
Abstract: Fluorescence microscopy is a key method in the life sciences. State of the art -omics methods combine fluorescence microscopy with complex protocols to visualize tens to thousands of features in each of millions of pixels across s les. These -omics methods require precise control of temperature, reagent application, and image acquisition parameters during iterative chemistry and imaging cycles conducted over the course of days or weeks. Automated execution of such methods enables robust and reproducible data generation. However, few commercial solutions exist for temperature controlled, fluidics coupled fluorescence imaging, and implementation of bespoke instrumentation requires specialized engineering expertise. Here we present PySeq2500, an open source Python code base and flow cell design that converts the Illumina HiSeq 2500 instrument into an open platform for programmable applications. Customizable PySeq2500 protocols enable experimental designs involving simultaneous 4-channel image acquisition, temperature control, reagent exchange, stable positioning, and s le integrity over extended experiments. To demonstrate accessible automation of complex, multi-day workflows, we use the PySeq2500 system for unattended execution of iterative indirect immunofluorescence imaging (4i). Our automated 4i method uses off-the-shelf antibodies over multiple cycles of staining, imaging, and antibody elution to build highly multiplexed maps of cell types and pathological features in mouse and postmortem human spinal cord sections. As demonstrated here, PySeq2500 enables non-specialists to develop and implement state of the art fluidics coupled imaging methods in a widely available benchtop system.
Publisher: eLife Sciences Publications, Ltd
Date: 14-05-2012
Publisher: Elsevier BV
Date: 03-2013
Publisher: Springer Science and Business Media LLC
Date: 22-12-2022
Publisher: The Company of Biologists
Date: 2016
DOI: 10.1242/DEV.141978
Abstract: Drosophila Elav is the founding member of the conserved family of Hu RNA binding proteins (RBPs), which collectively play critical and erse roles in post-transcriptional regulation. In particular, Elav has for & years served as the canonical neuronal marker, owing to the availability of specific monoclonal antibodies. Surprisingly, although Elav has a well-characterized neural cis-regulatory module, we find endogenous Elav is also ubiquitously transcribed and post-transcriptionally repressed in non-neural settings. In particular, mutant clones of multiple miRNA pathway components derepress ubiquitous Elav protein. Our re-annotation of the elav transcription unit shows that not only does it generate extended 3' UTR isoforms, its universal 3' UTR isoform is much longer than previously believed. This longer common 3' UTR region includes multiple conserved, high-affinity sites for the miR-279/996 family. Notably, out of several miRNA mutants tested, we find that endogenous Elav and a transgenic elav 3' UTR sensor are derepressed in mutant clones of mir-279/996. We also observe cross-repression of Elav by another RBP derepressed in non-neural miRNA pathway clones, namely Mei-P26. Finally, we demonstrate that ubiquitous Elav has regulatory capacity, since derepressed Elav can stabilize an Elav-responsive sensor. It is critical to restrict Elav outside of the nervous system as misexpression of Elav in non-neural territories has profoundly adverse consequences. Altogether, we define unexpected post-transcriptional mechanisms that direct appropriate cell-type specific expression of a conserved neural RBP.
Publisher: Springer Science and Business Media LLC
Date: 03-06-2021
Publisher: Springer Science and Business Media LLC
Date: 31-07-2017
DOI: 10.1038/NMETH.4380
Publisher: Elsevier
Date: 2012
Publisher: Springer Science and Business Media LLC
Date: 29-06-2023
Publisher: Cold Spring Harbor Laboratory
Date: 08-09-2020
DOI: 10.1101/2020.09.08.286914
Abstract: Recent technological advances have enabled massively parallel chromatin profiling with s ingle- c ell A ssay for T ransposase A ccessible C hromatin by seq uencing (scATAC-seq) in thousands of in idual cells. Here, we extend these approaches and present A TAC with S elect A ntigen P rofiling by seq uencing, ASAP-seq, a tool to simultaneously profile accessible chromatin and protein levels in thousands of single cells. Our approach pairs sparse scATAC-seq data with robust detection of hundreds of cell surface and intracellular protein markers and optional capture of mitochondrial DNA (mtDNA) for clonal tracking, thus concomitantly capturing three distinct modalities in single cells. Importantly, ASAP-seq uses a novel bridging approach that repurposes antibody:oligo conjugates designed for existing technologies that pair protein measurements with single cell RNA-seq. We demonstrate the utility of ASAP-seq by revealing coordinated and distinct changes in chromatin, RNA, and surface proteins during native hematopoietic differentiation, peripheral blood mononuclear cell stimulation, and as a combinatorial decoder and reporter of multiplexed perturbations in primary T cells.
Publisher: Cold Spring Harbor Laboratory
Date: 08-11-2018
DOI: 10.1101/466466
Abstract: Rapid technological progress in the recent years has allowed the high-throughput interrogation of different types of biomolecules from single cells. Combining several of these readouts into integrated multi-omic assays is essential to comprehensively understand and model cellular processes. Here, we report the development of E xpanded C RISPR-compatible C ellular I ndexing of T ranscriptomes and E pitopes by seq uencing (ECCITE-seq) for the high-throughput characterization of at least five modalities of information from each single cell: transcriptome, immune receptor clonotypes, surface markers, s le identity and sgRNAs. We demonstrate the use of ECCITE-seq to directly and efficiently capture sgRNA molecules and measure their effects on gene expression and protein levels, opening the possibility of performing high throughput single cell CRISPR screens with multimodal readout using existing libraries and commonly used vectors. Finally, by utilizing the combined phenotyping of clonotype and cell surface markers in immune cells, we apply ECCITE to study a lymphoma s le to discriminate cells and define molecular signatures of malignant cells within a heterogeneous population.
Publisher: Cold Spring Harbor Laboratory
Date: 28-06-2020
DOI: 10.1101/2020.06.28.175596
Abstract: The expression of inhibitory immune checkpoint molecules such as PD-L1 is frequently observed in human cancers and can lead to the suppression of T cell-mediated immune responses. Here we apply ECCITE-seq, a technology which combines pooled CRISPR screens with single-cell mRNA and surface protein measurements, to explore the molecular networks that regulate PD-L1 expression. We also develop a computational framework, mixscape , that substantially improves the signal-to-noise ratio in single-cell perturbation screens by identifying and removing confounding sources of variation. Applying these tools, we identify and validate regulators of PD-L1 , and leverage our multi-modal data to identify both transcriptional and post-transcriptional modes of regulation. In particular, we discover that the kelch-like protein KEAP1 and the transcriptional activator NRF2 , mediate levels of PD-L1 upregulation after IFNγ stimulation. Our results identify a novel mechanism for the regulation of immune checkpoints and present a powerful analytical framework for the analysis of multi-modal single-cell perturbation screens.
Publisher: Cold Spring Harbor Laboratory
Date: 20-11-2020
DOI: 10.1101/2020.11.20.390971
Abstract: Pooled CRISPR screens have been used to identify genes responsible for specific phenotypes and diseases, and, more recently, to connect genetic perturbations with multi-dimensional gene expression profiles. Here, we describe a method to link genome-wide chromatin accessibility to genetic perturbations in single cells. This scalable, cost-effective method combines pooled CRISPR perturbations with a single-cell combinatorial indexing assay for transposase-accessible chromatin (CRISPR-sciATAC). Using a human and mouse species-mixing experiment, we show that CRISPR-sciATAC separates single cells with a low doublet rate. Then, in human myelogenous leukemia cells, we apply CRISPR-sciATAC to target 21 chromatin-related genes that are frequently mutated in cancer and 84 subunits and cofactors of chromatin remodeling complexes, generating chromatin accessibility data for ~30,000 single cells. Using this large-scale atlas, we correlate loss of specific chromatin remodelers with changes in accessibility — globally and at the binding sites of in idual transcription factors. For ex le, we show that loss of the H3K27 methyltransferase EZH2 leads to increased accessibility at heterochromatic regions involved in embryonic development and triggers expression of multiple genes in the HOXA and HOXD clusters. At a subset of regulatory sites, we also analyze dynamic changes in nucleosome spacing upon loss of chromatin remodelers. CRISPR-sciATAC is a high-throughput, low-cost single-cell method that can be applied broadly to study the role of genetic perturbations on chromatin in normal and disease states.
Publisher: eLife Sciences Publications, Ltd
Date: 16-04-2021
DOI: 10.7554/ELIFE.61973
Abstract: Simultaneous measurement of surface proteins and gene expression within single cells using oligo-conjugated antibodies offers high-resolution snapshots of complex cell populations. Signal from oligo-conjugated antibodies is quantified by high-throughput sequencing and is highly scalable and sensitive. We investigated the response of oligo-conjugated antibodies towards four variables: concentration, staining volume, cell number at staining, and tissue. We find that staining with recommended antibody concentrations causes unnecessarily high background and amount of antibody used can be drastically reduced without loss of biological information. Reducing staining volume only affects antibodies targeting abundant epitopes used at low concentrations and is counteracted by reducing cell numbers. Adjusting concentrations increases signal, lowers background, and reduces costs. Background signal can account for a major fraction of total sequencing and is primarily derived from antibodies used at high concentrations. This study provides new insight into titration response and background of oligo-conjugated antibodies and offers concrete guidelines to improve such panels.
Publisher: Cold Spring Harbor Laboratory
Date: 12-10-2020
DOI: 10.1101/2020.10.12.335331
Abstract: The simultaneous measurement of multiple modalities, known as multimodal analysis, represents an exciting frontier for single-cell genomics and necessitates new computational methods that can define cellular states based on multiple data types. Here, we introduce ‘weighted-nearest neighbor’ analysis, an unsupervised framework to learn the relative utility of each data type in each cell, enabling an integrative analysis of multiple modalities. We apply our procedure to a CITE-seq dataset of hundreds of thousands of human white blood cells alongside a panel of 228 antibodies to construct a multimodal reference atlas of the circulating immune system. We demonstrate that integrative analysis substantially improves our ability to resolve cell states and validate the presence of previously unreported lymphoid subpopulations. Moreover, we demonstrate how to leverage this reference to rapidly map new datasets, and to interpret immune responses to vaccination and COVID-19. Our approach represents a broadly applicable strategy to analyze single-cell multimodal datasets, including paired measurements of RNA and chromatin state, and to look beyond the transcriptome towards a unified and multimodal definition of cellular identity. Installation instructions, documentation, tutorials, and CITE-seq datasets are available at eurat
Publisher: Elsevier BV
Date: 03-2012
Publisher: Elsevier BV
Date: 03-2022
Publisher: Cold Spring Harbor Laboratory
Date: 29-10-2015
Abstract: N 6 -threonylcarbamoyl-adenosine (t6A) is one of the few RNA modifications that is universally present in life. This modification occurs at high frequency at position 37 of most tRNAs that decode ANN codons, and stabilizes cognate anticodon–codon interactions. Nearly all genetic studies of the t6A pathway have focused on single-celled organisms. In this study, we report the isolation of an extensive allelic series in the Drosophila ortholog of the core t6A biosynthesis factor Kae1. kae1 hemizygous larvae exhibit decreases in t6A that correlate with allele strength however, we still detect substantial t6A-modified tRNAs even during the extended larval phase of null alleles. Nevertheless, complementation of Drosophila Kae1 and other t6A factors in corresponding yeast null mutants demonstrates that these metazoan genes execute t6A synthesis. Turning to the biological consequences of t6A loss, we characterize prominent kae1 melanotic masses and show that they are associated with lymph gland overgrowth and ectopic generation of lamellocytes. On the other hand, kae1 mutants exhibit other phenotypes that reflect insufficient tissue growth. Interestingly, whole-tissue and clonal analyses show that strongly mitotic tissues such as imaginal discs are exquisitely sensitive to loss of kae1 , whereas nonproliferating tissues are less affected. Indeed, despite overt requirements of t6A for growth of many tissues, certain strong kae1 alleles achieve and sustain enlarged body size during their extended larval phase. Our studies highlight tissue-specific requirements of the t6A pathway in a metazoan context and provide insights into the erse biological roles of this fundamental RNA modification during animal development and disease.
Publisher: Elsevier BV
Date: 05-2012
Publisher: Oxford University Press (OUP)
Date: 12-11-2013
DOI: 10.1093/NAR/GKT1038
Publisher: Cold Spring Harbor Laboratory
Date: 02-03-2017
DOI: 10.1101/113068
Abstract: Recent high-throughput single-cell sequencing approaches have been transformative for understanding complex cell populations, but are unable to provide additional phenotypic information, such as protein levels of cell-surface markers. Using oligonucleotide-labeled antibodies, we integrate measurements of cellular proteins and transcriptomes into an efficient, sequencing-based readout of single cells. This method is compatible with existing single-cell sequencing approaches and will readily scale as the throughput of these methods increase.
Publisher: ZappyLab, Inc.
Date: 26-02-2018
DOI: 10.17504/PROTOCOLS.IO.NGZDBX6
Abstract: This collection contains our main protocols for performing CITE-seq and Cell Hashing, specifically on Drop-seq or10x Genomics single cell 3′ v2 chemistry. CITE-seq: Cellular Indexing of Transcriptomes and Epitopes by Sequencing (CITE-seq) is a multimodal single cell phenotyping method developed in theTechnology Innovation labat the New York Genome Center in collaboration with the Satija lab. CITE-seq uses DNA-barcoded antibodies to convert detection of proteins into a quantitative, sequenceable readout. Antibody-bound oligos act as synthetic transcripts that are captured during most large-scale oligodT-based scRNA-seq library preparation protocols (e.g. 10x Genomics, Drop-seq, ddSeq). This allows for immunophenotyping of cells with a potentially limitless number of markers and unbiased transcriptome analysis using existing single-cell sequencing approaches. Cell Hashing: S le multiplexing and super-loading onsingle cellRNA-sequencing platforms. Cell Hashinguses a series of oligo-tagged antibodies against ubiquitously expressed surface proteins with different barcodes to uniquely label cells from distinct s les, which can be subsequently pooled in onescRNA-seq run. By sequencing these tags alongside the cellular transcriptome, we can assign each cell to its s le of origin, and robustly identify doublets originating from multiple s les.
Publisher: Elsevier BV
Date: 03-2020
Publisher: Springer Science and Business Media LLC
Date: 03-2021
Publisher: Elsevier BV
Date: 02-2010
Publisher: American Society of Hematology
Date: 21-10-2021
Abstract: Cutaneous T-cell lymphoma (CTCL) is a heterogeneous group of mature T-cell neoplasms characterized by the accumulation of clonal malignant CD4+ T cells in the skin. The most common variant of CTCL, mycosis fungoides (MF ), is confined to the skin in early stages but can be accompanied by extracutaneous dissemination of malignant T cells to the blood and lymph nodes in advanced stages of disease. Sézary syndrome (SS), a leukemic form of disease, is characterized by significant blood involvement. Little is known about the transcriptional and genomic relationship between skin- and blood-residing malignant T cells in CTCL. To identify and interrogate malignant clones in matched skin and blood from patients with leukemic MF and SS, we combine T-cell receptor clonotyping with quantification of gene expression and cell surface markers at the single cell level. Our data reveal clonal evolution at a transcriptional and genetic level within the malignant populations of in idual patients. We highlight highly consistent transcriptional signatures delineating skin- and blood-derived malignant T cells. Analysis of these 2 populations suggests that environmental cues, along with genetic aberrations, contribute to transcriptional profiles of malignant T cells. Our findings indicate that the skin microenvironment in CTCL promotes a transcriptional response supporting rapid malignant expansion, as opposed to the quiescent state observed in the blood, potentially influencing efficacy of therapies. These results provide insight into tissue-specific characteristics of cancerous cells and underscore the need to address the patients’ in idual malignant profiles at the time of therapy to eliminate all subclones.
Publisher: Cold Spring Harbor Laboratory
Date: 06-2020
DOI: 10.1101/2020.05.31.126763
Abstract: Many methods exist to detect RNA modifications by short-read sequencing, relying on either antibody enrichment of transcripts bearing modified bases or mutational profiling approaches which require conversion to cDNA. Endogenous modifications are present on several major classes of RNA including tRNA, rRNA and mRNA and can modulate erse biological processes such as genetic recoding, mRNA export and RNA folding. In addition, exogenous modifications can be introduced to RNA molecules to reveal RNA structure and dynamics. Limitations on read length and library size inherent in short-read-based methods dissociate modifications from their native context, preventing single molecule analysis and modification phasing. Here we demonstrate direct RNA nanopore sequencing to detect endogenous and exogenous RNA modifications over long sequence distance at the single molecule level. We demonstrate comprehensive detection of endogenous modifications in E. coli and S. cerevisiae ribosomal RNA (rRNA) using current signal deviations. Notably 2’-O-methyl (Nm) modifications generated a discernible shift in current signal and event level dwell times. We show that dwell times are mediated by the RNA motor protein which sits atop the nanopore. Further, we characterize a recently described small adduct-generating 2’-O-acylation reagent, acetylimidazole (AcIm) for exogenously labeling flexible nucleotides in RNA. Finally, we demonstrate the utility of AcIm for single molecule RNA structural probing using nanopore sequencing.
Publisher: Cold Spring Harbor Laboratory
Date: 16-06-2020
DOI: 10.1101/2020.06.15.153080
Abstract: Simultaneous measurement of surface proteins and gene expression within single cells using oligo-conjugated antibodies offers high resolution snapshots of complex cell populations. Signal from oligo-conjugated antibodies is quantified by high-throughput sequencing and is highly scalable and sensitive. In this study, we investigated the response of oligo-conjugated antibodies towards four variables: Concentration, staining volume, cell number at staining, and tissue. We find that staining with recommended antibody concentrations cause unnecessarily high background and that concentrations can be drastically reduced without loss of biological information. Reducing volume only affects antibodies targeting abundant epitopes used at low concentrations and is counteracted by reducing cell numbers. Adjusting concentrations increases signal, lowers background and reduces costs. Background signal can account for a major fraction of the total sequencing and is primarily derived from antibodies used at high concentrations. Together, this study provides new insight into the titration response and background of oligo-conjugated antibodies and offers concrete guidelines on how such panels can be improved. Oligo-conjugated antibodies are a powerful tool but require thorough optimization to reduce background signal, increase sensitivity, and reduce sequencing costs.
Publisher: Cold Spring Harbor Laboratory
Date: 14-09-2021
DOI: 10.1101/2021.09.13.460120
Abstract: New technologies that profile chromatin modifications at single-cell resolution offer enormous promise for functional genomic characterization. However, the sparsity of these measurements and the challenge of integrating multiple binding maps represent significant challenges. Here we introduce scCUT& Tag-pro, a multimodal assay for profiling protein-DNA interactions coupled with the abundance of surface proteins in single cells. In addition, we introduce scChromHMM, which integrates data from multiple experiments to infer and annotate chromatin states based on combinatorial histone modification patterns. We apply these tools to perform an integrated analysis across nine different molecular modalities in circulating human immune cells. We demonstrate how these two approaches can characterize dynamic changes in the function of in idual genomic elements across both discrete cell states and continuous developmental trajectories, nominate associated motifs and regulators that establish chromatin states, and identify extensive and cell type-specific regulatory priming. Finally, we demonstrate how our integrated reference can serve as a scaffold to map and improve the interpretation of additional scCUT& Tag datasets.
Publisher: Elsevier BV
Date: 09-2010
Publisher: Informa UK Limited
Date: 02-2009
DOI: 10.1128/MCB.01524-08
Publisher: Springer Science and Business Media LLC
Date: 26-05-2013
DOI: 10.1038/NSMB.2606
Publisher: Cold Spring Harbor Laboratory
Date: 11-05-2022
DOI: 10.1101/2022.05.11.491515
Abstract: In normal somatic tissue differentiation, changes in chromatin accessibility govern priming and commitment of precursors towards cellular fates. In turn, somatic mutations can disrupt differentiation topologies leading to abnormal clonal outgrowth. However, defining the impact of somatic mutations on the epigenome in human s les is challenging due to admixed mutated and wildtype cells. To chart how somatic mutations disrupt epigenetic landscapes in human clonal outgrowths, we developed Genotyping of Targeted loci with single-cell Chromatin Accessibility (GoT-ChA). This high-throughput, broadly accessible platform links genotypes to chromatin accessibility at single-cell resolution, across thousands of cells within a single assay. We applied GoT-ChA to CD34 + cells from myeloproliferative neoplasm (MPN) patients with JAK2 V617F -mutated hematopoiesis, where the JAK2 mutation is known to perturb hematopoietic differentiation. Differential accessibility analysis between wildtype and JAK2 V617F mutant progenitors revealed both cell-intrinsic and cell state-specific shifts within mutant hematopoietic precursors. An early subset of mutant hematopoietic stem and progenitor cells (HSPCs) exhibited a cell-intrinsic pro-inflammatory signature characterized by increased NF-κB and JUN/FOS transcription factor motif accessibility. In addition, mutant HSPCs showed increased myeloid/erythroid epigenetic priming, preceding increased erythroid and megakaryocytic cellular output. Erythroid progenitors displayed aberrant regulation of the γ-globin locus, providing an intrinsic epigenetic basis for the dysregulated fetal hemoglobin expression observed in MPNs. In contrast, megakaryocytic progenitors exhibited a more specialized inflammatory chromatin landscape relative to early HSPCs, with increased accessibility of pro-fibrotic JUN/FOS transcription factors. Notably, analysis of myelofibrosis patients treated with JAK inhibitors revealed an overall loss of mutant-specific phenotypes without modifying clonal burden, consistent with clinical responses. Finally, expansion of the multi-modality capability of GoT-ChA to integrate mitochondrial genome profiling and cell surface protein expression measurement enabled genotyping imputation and discovery of aberrant cellular phenotypes. Collectively, we show that the JAK2 V617F mutation leads to epigenetic rewiring in a cell-intrinsic and cell type-specific manner. We envision that GoT-ChA will thus serve as a foundation for broad future explorations to uncover the critical link between mutated somatic genotypes and epigenetic alterations across clonal populations in malignant and non-malignant contexts.
Publisher: American Association for the Advancement of Science (AAAS)
Date: 19-05-2023
Abstract: Most variants associated with complex traits and diseases identified by genome-wide association studies (GWAS) map to noncoding regions of the genome with unknown effects. Using ancestrally erse, biobank-scale GWAS data, massively parallel CRISPR screens, and single-cell transcriptomic and proteomic sequencing, we discovered 124 cis -target genes of 91 noncoding blood trait GWAS loci. Using precise variant insertion through base editing, we connected specific variants with gene expression changes. We also identified trans -effect networks of noncoding loci when cis target genes encoded transcription factors or microRNAs. Networks were themselves enriched for GWAS variants and demonstrated polygenic contributions to complex traits. This platform enables massively parallel characterization of the target genes and mechanisms of human noncoding variants in both cis and trans .
Publisher: Thomas Telford Ltd.
Date: 09-2006
Publisher: Elsevier BV
Date: 05-2007
DOI: 10.1016/J.TIG.2007.03.007
Abstract: The generation and analysis of mutants is central to studies of gene function in model organisms. Methods for random mutagenesis in Drosophila melanogaster have been available for many years, but an alternative approach--targeted mutagenesis using homologous recombination--has only recently been developed. This approach has the advantage of specificity, because genes of interest can be altered. One might expect with a gene-targeting approach that the frequency of background mutations would be minimal. Unfortunately, we have found that this is not the case. Although the possibility of background mutations arising during homologous-recombination-based gene targeting has been raised in the literature, it is not routinely taken into account when using this technique. Our experience suggests that it can be a considerable problem but that it has a relatively simple solution.
Publisher: The Company of Biologists
Date: 15-07-2013
DOI: 10.1242/DEV.092817
Abstract: MicroRNAs (miRNAs) are regulators of global gene expression and function in a broad range of biological processes. Recent studies have suggested that miRNAs can function as tumor suppressors or oncogenes by modulating the activities of evolutionarily conserved signaling pathways that are commonly dysregulated in cancer. We report the identification of the miR-310 to miR-313 (miR-310/13) cluster as a novel antagonist of Wingless (Drosophila Wnt) pathway activity in a functional screen for Drosophila miRNAs. We demonstrate that miR-310/13 can modulate Armadillo (Arm Drosophila β-catenin) expression and activity by directly targeting the 3′-UTRs of arm and pangolin (Drosophila TCF) in vivo. Notably, the miR-310/13-deficient flies exhibit abnormal germ and somatic cell differentiation in the male gonad, which can be rescued by reducing Arm protein levels or activity. Our results implicate a previously unrecognized function for miR-310/13 in d ening the activity of Arm in early somatic and germline progenitor cells, whereby inappropriate/sustained activation of Arm-mediated signaling or cell adhesion may impact normal differentiation in the Drosophila male gonad.
Publisher: Cold Spring Harbor Laboratory
Date: 02-11-2018
DOI: 10.1101/460147
Abstract: Single cell transcriptomics (scRNA-seq) has transformed our ability to discover and annotate cell types and states, but deep biological understanding requires more than a taxonomic listing of clusters. As new methods arise to measure distinct cellular modalities, including high-dimensional immunophenotypes, chromatin accessibility, and spatial positioning, a key analytical challenge is to integrate these datasets into a harmonized atlas that can be used to better understand cellular identity and function. Here, we develop a computational strategy to “anchor” erse datasets together, enabling us to integrate and compare single cell measurements not only across scRNA-seq technologies, but different modalities as well. After demonstrating substantial improvement over existing methods for data integration, we anchor scRNA-seq experiments with scATAC-seq datasets to explore chromatin differences in closely related interneuron subsets, and project single cell protein measurements onto a human bone marrow atlas to annotate and characterize lymphocyte populations. Lastly, we demonstrate how anchoring can harmonize in-situ gene expression and scRNA-seq datasets, allowing for the transcriptome-wide imputation of spatial gene expression patterns, and the identification of spatial relationships between mapped cell types in the visual cortex. Our work presents a strategy for comprehensive integration of single cell data, including the assembly of harmonized references, and the transfer of information across datasets. Availability: Installation instructions, documentation, and tutorials are available at: eurat
Publisher: Springer Science and Business Media LLC
Date: 24-03-2022
Publisher: Springer Science and Business Media LLC
Date: 22-04-2019
Publisher: Cold Spring Harbor Laboratory
Date: 21-12-2017
DOI: 10.1101/237693
Abstract: Despite rapid developments in single cell sequencing technology, s le-specific batch effects, detection of cell doublets, and the cost of generating massive datasets remain outstanding challenges. Here, we introduce cell “hashing”, where oligo-tagged antibodies against ubiquitously expressed surface proteins are used to uniquely label cells from distinct s les, which can be subsequently pooled. By sequencing these tags alongside the cellular transcriptome, we can assign each cell to its s le of origin, and robustly identify doublets originating from multiple s les. We demonstrate our approach by pooling eight human PBMC s les on a single run of the 10x Chromium system, substantially reducing our per-cell costs for library generation. Cell “hashing” is inspired by, and complementary to, elegant multiplexing strategies based on genetic variation, which we also leverage to validate our results. We therefore envision that our approach will help to generalize the benefits of single cell multiplexing to erse s les and experimental designs.
Publisher: Cold Spring Harbor Laboratory
Date: 21-04-2021
DOI: 10.1101/2021.04.20.21255677
Abstract: SARS-CoV-2 infection and vaccination elicit potent immune responses. Our study presents a comprehensive multimodal single-cell dataset of peripheral blood of patients with acute COVID-19 and of healthy volunteers before and after receiving the SARS-CoV-2 mRNA vaccine and booster. We compared host immune responses to the virus and vaccine using transcriptional profiling, coupled with B/T cell receptor repertoire reconstruction. COVID-19 patients displayed an enhanced interferon signature and cytotoxic gene upregulation, absent in vaccine recipients. These findings were validated in an independent dataset. Analysis of B and T cell repertoires revealed that, while the majority of clonal lymphocytes in COVID-19 patients were effector cells, clonal expansion was more evident among circulating memory cells in vaccine recipients. Furthermore, while clonal αβ T cell responses were observed in both COVID-19 patients and vaccine recipients, dramatic expansion of clonal γδT cells was found only in infected in iduals. Our dataset enables comparative analyses of immune responses to infection versus vaccination, including clonal B and T cell responses. Integrating our data with publicly available datasets allowed us to validate our findings in larger cohorts. To our knowledge, this is the first dataset to include comprehensive profiling of longitudinal s les from healthy volunteers pre ost SARS-CoV-2 vaccine and booster.
Publisher: Springer Science and Business Media LLC
Date: 16-03-2022
DOI: 10.1038/S41586-022-04494-7
Abstract: The engineering of autologous patient T cells for adoptive cell therapies has revolutionized the treatment of several types of cancer
Publisher: Springer Science and Business Media LLC
Date: 20-04-2020
DOI: 10.1038/S41467-020-15710-1
Abstract: Peptide exchange technologies are essential for the generation of pMHC-multimer libraries used to probe erse, polyclonal TCR repertoires in various settings. Here, using the molecular chaperone TAPBPR, we develop a robust method for the capture of stable, empty MHC-I molecules comprising murine H2 and human HLA alleles, which can be readily tetramerized and loaded with peptides of choice in a high-throughput manner. Alternatively, catalytic amounts of TAPBPR can be used to exchange placeholder peptides with high affinity peptides of interest. Using the same system, we describe high throughput assays to validate binding of multiple candidate peptides on empty MHC-I/TAPBPR complexes. Combined with tetramer-barcoding via a multi-modal cellular indexing technology, ECCITE-seq, our approach allows a combined analysis of TCR repertoires and other T cell transcription profiles together with their cognate antigen specificities in a single experiment. The new approach allows TCR MHC interactions to be interrogated easily at large scale.
Publisher: American Association for the Advancement of Science (AAAS)
Date: 03-05-2019
Abstract: A novel Drosophila -based approach resulted in a significant response by a patient with KRAS-mutant colorectal cancer.
Publisher: Cold Spring Harbor Laboratory
Date: 08-04-2021
DOI: 10.1101/2021.04.07.438882
Abstract: The majority of variants associated with complex traits and common diseases identified by genome-wide association studies (GWAS) map to noncoding regions of the genome with unknown regulatory effects in cis and trans . By leveraging biobank-scale GWAS data, massively parallel CRISPR screens and single cell transcriptome sequencing, we discovered target genes of noncoding variants for blood trait loci. The closest gene was often the target gene, but this was not always the case. We also identified trans -effects networks of noncoding variants when cis target genes encoded transcription factors, such as GFI1B and NFE2 . We observed that GFI1B trans -target genes were enriched for GFI1B binding sites and fine-mapped GWAS variants, and expressed in human bone marrow progenitor cells, suggesting that GFI1B acts as a master regulator of blood traits. This platform will enable massively parallel assays to catalog the target genes of human noncoding variants in both cis and trans .
Publisher: Cold Spring Harbor Laboratory
Date: 23-09-2011
DOI: 10.1261/RNA.2983511
Abstract: Canonical animal microRNAs (miRNAs) are ∼22-nt regulatory RNAs generated by stepwise cleavage of primary hairpin transcripts by the Drosha and Dicer RNase III enzymes. We performed a genetic screen using an miRNA-repressed reporter in the Drosophila eye and recovered the first reported alleles of fly drosha , an allelic series of its dsRBD partner pasha , and novel alleles of dicer-1 . Analysis of drosha mutants provided direct confirmation that mirtrons are independent of this nuclease, as inferred earlier from pasha knockouts. We further used these mutants to demonstrate in vivo cross-regulation of Drosha and Pasha in the intact animal, confirming remarkable conservation of a homeostatic mechanism that aligns their respective levels. Although the loss of core miRNA pathway components is universally lethal in animals, we unexpectedly recovered hypomorphic alleles that gave adult escapers with overtly normal development. However, the mutant photoreceptor neurons exhibited reduced synaptic transmission, without accompanying defects in neuronal development or maintenance. These findings indicate that synaptic function is especially sensitive to optimal miRNA pathway function. These allelic series of miRNA pathway mutants should find broad usage in studies of miRNA biogenesis and biology in the Drosophila system.
Publisher: Cold Spring Harbor Laboratory
Date: 28-09-2020
DOI: 10.1101/2020.09.27.313445
Abstract: Although degree of T-cell infiltration in CRC was shown to correlate with a positive prognosis, the contribution of phenotypically and functionally distinct T cell subtypes within tumors remains unclear. We analyzed 37,931 single T cells with respect to transcriptome, TCR sequence and 23 cell surface proteins, from tumors and adjacent normal colon of 16 patients. Our comprehensive analysis revealed two phenotypically distinct cytotoxic T cell populations within tumors, including positively prognostic effector memory cells and non-prognostic resident memory cells. These cytotoxic T cell infiltrates transitioned from effector memory to resident memory in a stage-dependent manner. We further defined several Treg subpopulations within tumors. While Tregs overall were associated with positive clinical outcomes, CD38 + peripherally-derived Tregs, phenotypically related to Th17 cells, correlated with poor outcomes independent of cancer stage. Thus, our data highlight the ersity of T cells in CRC and demonstrate the prognostic significance of distinct T cell subtypes, which could inform therapeutic strategies.
Publisher: Springer Science and Business Media LLC
Date: 29-04-2021
Location: United States of America
No related grants have been discovered for Peter Smibert.