HuBMAP Publications

There are 90 publications.
Publish DateTitleAbstractAuthor(s)HuBMAP Component
2018-10-29Identification of spatially associated subpopulations by combining scRNAseq and sequential fluorescence in situ hybridization dataZhu Q, Shah S, Dries R, Cai L, Yuan GC.TTD-Cal TechHow intrinsic gene-regulatory networks interact with a cell's spatial environment to define its identity remains poorly understood. We developed an approach to distinguish between intrinsic and extrinsic effects on global gene expression by integrating analysis of sequencing-based and imaging-based single-cell transcriptomic profiles, using cross-platform cell type mapping combined with a hidden Markov random field model. We applied this approach to dissect the cell-type- and spatial-domain-associated heterogeneity in the mouse visual cortex region. Our analysis identified distinct spatially associated, cell-type-independent signatures in the glutamatergic and astrocyte cell compartments. Using these signatures to analyze single-cell RNA sequencing data, we identified previously unknown spatially associated subpopulations, which were validated by comparison with anatomical structures and Allen Brain Atlas images.
2018-12-11Forecasting innovations in science, technology, and educationBörner K, Rouse WB, Trunfio P, Stanley HE.HIVE MC-IUHuman survival depends on our ability to predict future outcomes so that we can make informed decisions. Human cognition and perception are optimized for local, short-term decision-making, such as deciding when to fight or flight, whom to mate, or what to eat. For more elaborate decisions (e.g., when to harvest, when to go to war or not, and whom to marry), people used to consult oracles—prophetic predictions of the future inspired by the gods. Over time, oracles were replaced by models of the structure and dynamics of natural, technological, and social systems. In the 21st century, computational models and visualizations of model results inform much of our decision-making: near real-time weather forecasts help us decide when to take an umbrella, plant, or harvest; where to ground airplanes; or when to evacuate inhabitants in the path of a hurricane, tornado, or flood. Long-term weather and climate forecasts predict a future with increasing torrential rains, stronger winds, and more frequent drought, landslides, and forest fires as well as rising sea levels, enabling decision makers to prepare for these changes by building dikes, moving cities and roads, and building larger water reservoirs and better storm sewers.
2018-12-19Cell Hashing with barcoded antibodies enables multiplexing and doublet detection for single cell genomicsStoeckius M, Zheng S, Houck-Loomis B, Hao S, Yeung BZ, Mauck WM, Smibert P, Satija R.HIVE MC-NYGCDespite rapid developments in single cell sequencing, sample-specific batch effects, detection of cell multiplets, and experimental costs remain outstanding challenges. Here, we introduce Cell Hashing, where oligo-tagged antibodies against ubiquitously expressed surface proteins uniquely label cells from distinct samples, which can be subsequently pooled. By sequencing these tags alongside the cellular transcriptome, we can assign each cell to its original sample, robustly identify cross-sample multiplets, and “super-load” commercial droplet-based systems for significant cost reduction. We validate our approach using a complementary genetic approach and demonstrate how hashing can generalize the benefits of single cell multiplexing to diverse samples and experimental designs.
2019-02-01Protein identification strategies in MALDI imaging mass spectrometry: a brief reviewRyan DJ, Spraggins JM, Caprioli RM.TMC-Vanderbilt (Kidney)Matrix assisted laser desorption/ionization (MALDI) imaging mass spectrometry (IMS) is a powerful technology used to investigate the spatial distributions of thousands of molecules throughout a tissue section from a single experiment. As proteins represent an important group of functional molecules in tissue and cells, the imaging of proteins has been an important point of focus in the development of IMS technologies and methods. Protein identification is crucial for the biological contextualization of molecular imaging data. However, gas-phase fragmentation efficiency of MALDI generated proteins presents significant challenges, making protein identification directly from tissue difficult. This review highlights methods and technologies specifically related to protein identification that have been developed to overcome these challenges in MALDI IMS experiments.
2019-02-05Data visualization literacy: Definitions, conceptual frameworks, exercises, and assessmentsBörner K, Bueckle A and Ginda M.HIVE MC-IUIn the information age, the ability to read and construct data visualizations becomes as important as the ability to read and write text. However, while standard definitions and theoretical frameworks to teach and assess textual, mathematical, and visual literacy exist, current data visualization literacy (DVL) definitions and frameworks are not comprehensive enough to guide the design of DVL teaching and assessment. This paper introduces a data visualization literacy framework (DVL-FW) that was specifically developed to define, teach, and assess DVL. The holistic DVL-FW promotes both the reading and construction of data visualizations, a pairing analogous to that of both reading and writing in textual literacy and understanding and applying in mathematical literacy. Specifically, the DVL-FW defines a hierarchical typology of core concepts and details the process steps that are required to extract insights from data. Advancing the state of the art, the DVL-FW interlinks theoretical and procedural knowledge and showcases how both can be combined to design curricula and assessment measures for DVL. Earlier versions of the DVL-FW have been used to teach DVL to more than 8,500 residential and online students, and results from this effort have helped revise and validate the DVL-FW presented here.
2019-02-15Dhaka: Variational Autoencoder for Unmasking Tumor Heterogeneity from Single Cell Genomic DataRashid S, Shah S, Bar-Joseph Z, Pandya R.HIVE TC-CMUMOTIVATION: Intra-tumor heterogeneity is one of the key confounding factors in deciphering tumor evolution. Malignant cells exhibit variations in their gene expression, copy numbers, and mutation even when originating from a single progenitor cell. Single cell sequencing of tumor cells has recently emerged as a viable option for unmasking the underlying tumor heterogeneity. However, extracting features from single cell genomic data in order to infer their evolutionary trajectory remains computationally challenging due to the extremely noisy and sparse nature of the data. RESULTS: Here we describe 'Dhaka', a variational autoencoder method which transforms single cell genomic data to a reduced dimension feature space that is more efficient in differentiating between (hidden) tumor subpopulations. Our method is general and can be applied to several different types of genomic data including copy number variation from scDNA-Seq and gene expression from scRNA-Seq experiments. We tested the method on synthetic and 6 single cell cancer datasets where the number of cells ranges from 250 to 6000 for each sample. Analysis of the resulting feature space revealed subpopulations of cells and their marker genes. The features are also able to infer the lineage and/or differentiation trajectory between cells greatly improving upon prior methods suggested for feature extraction and dimensionality reduction of such data. AVAILABILITY AND IMPLEMENTATION: All the datasets used in the paper are publicly available and developed software package and supporting info is available on Github
2019-02-20The single-cell transcriptional landscape of mammalian organogenesisCao J, Spielmann M, Qiu X, Huang X, Ibrahim DM, Hill AJ, Zhang F, Mundlos S, Christiansen L, Steemers FJ, Trapnell C & Shendure JTMC-Cal TechMammalian organogenesis is a remarkable process. Within a short timeframe, the cells of the three germ layers transform into an embryo that includes most of the major internal and external organs. Here we investigate the transcriptional dynamics of mouse organogenesis at single-cell resolution. Using single-cell combinatorial indexing, we profiled the transcriptomes of around 2 million cells derived from 61 embryos staged between 9.5 and 13.5 days of gestation, in a single experiment. The resulting ‘mouse organogenesis cell atlas’ (MOCA) provides a global view of developmental processes during this critical window. We use Monocle 3 to identify hundreds of cell types and 56 trajectories, many of which are detected only because of the depth of cellular coverage, and collectively define thousands of corresponding marker genes. We explore the dynamics of gene expression within cell types and trajectories over time, including focused analyses of the apical ectodermal ridge, limb mesenchyme and skeletal muscle.
2019-03-01Multiple TOF/TOF Events in a Single Laser Shot for Multiplexed Lipid Identifications in MALDI Imaging Mass SpectrometryPrentice BM, McMillen JC, Caprioli RMTMC-Vanderbilt (Kidney)Tandem mass spectrometry (MS/MS) is often used to identify lipids in matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI IMS) workflows. The molecular specificity afforded by MS/MS is crucial on MALDI time-of-flight (TOF) platforms that generally lack high resolution accurate mass measurement capabilities. Unfortunately, imaging MS/MS workflows generally only monitor a single precursor ion over the imaged area, limiting the throughput of this methodology. Herein, we demonstrate that multiple TOF/TOF events performed in each laser shot can be used to improve the throughput of imaging MS/MS. This is shown to enable the simultaneous identification of multiple phosphatidylcholine lipids in rat brain tissue. Uniquely, the separation in time achieved for the precursor ions in the TOF-1 region of the instrument is maintained for the fragment ions as they are analyzed in TOF-2, allowing for the differentiation of fragment ions of the exact same m/z derived from different precursor ions (e.g., the m/z 163 fragment ion from precursor ion m/z 772.5 is easily distinguished from the m/z 163 fragment ion from precursor ion m/z 826.5). This multiplexed imaging MS/MS approach allows for the acquisition of complete fragment ion spectra for multiple precursor ions per laser shot.
2019-03-25Transcriptome-scale super-resolved imaging in tissues by RNA seqFISHEng CL, Lawson M, Zhu Q, Dries R, Koulena N, Takei Y, Yun J, Cronin C, Karp C, Yuan GC, Cai L.TTD-Cal TechImaging the transcriptome in situ with high accuracy has been a major challenge in single-cell biology, which is particularly hindered by the limits of optical resolution and the density of transcripts in single cells. Here we demonstrate an evolution of sequential fluorescence in situ hybridization (seqFISH+). We show that seqFISH+ can image mRNAs for 10,000 genes in single cells-with high accuracy and sub-diffraction-limit resolution-in the cortex, subventricular zone and olfactory bulb of mouse brain, using a standard confocal microscope. The transcriptome-level profiling of seqFISH+ allows unbiased identification of cell classes and their spatial organization in tissues. In addition, seqFISH+ reveals subcellular mRNA localization patterns in cells and ligand-receptor pairs across neighbouring cells. This technology demonstrates the ability to generate spatial cell atlases and to perform discovery-driven studies of biological processes in situ.
2019-04-06Imaging mass spectrometry enables molecular profiling of mouse and human pancreatic tissuePrentice BM, Hart NJ, Phillips N, Haliyur R, Judd A, Armandala R, Spraggins JM, Lowe CL, Boyd KL, Stein RW, Wright CV, Norris JL, Powers AC, Brissova M, Caprioli RM.TMC-Vanderbilt (Kidney)The molecular response and function of pancreatic islet cells during metabolic stress is a complex process. The anatomical location and small size of pancreatic islets coupled with current methodological limitations have prevented the achievement of a complete, coherent picture of the role that lipids and proteins play in cellular processes under normal conditions and in diseased states. Herein, we describe the development of untargeted tissue imaging mass spectrometry (IMS) technologies for the study of in situ protein and, more specifically, lipid distributions in murine and human pancreases.
2019-04-19The Importance of Clinical Tissue ImagingSpraggins JM, Schwamborn K, Heeren RMA, Eberlin LS.TMC-Vanderbilt (Kidney)Tissue imaging by mass spectrometry (MS) combines the sensitivity and molecular specificity of MS with the spatial fidelity of classical histology for analysis of metabolites, lipids and proteins in tissues (Fig. 1). MS-based imaging is label-free, untargeted, sensitive, and specific, thereby enabling application in both basic biomedical research and the clinical laboratory. While all tissue imaging experiments are conceptually similar in their ability to generate spatial molecular data; ionization, data collection, and purpose vary widely. Here, we highlight recent technical advances and efforts that are motivating translational applications of this emerging technology.
2019-05-01SABER amplifies FISH: enhanced multiplexed imaging of RNA and DNA in cells and tissuesKishi JY, Lapan SW, Beliveau BJ, West ER, Zhu A, Sasaki HM, Saka SK, Wang Y, Cepko CL, Yin P.TTD-HarvardFluorescence in situ hybridization (FISH) reveals the abundance and positioning of nucleic acid sequences in fixed samples. Despite recent advances in multiplexed amplification of FISH signals, it remains challenging to achieve high levels of simultaneous amplification and sequential detection with high sampling efficiency and simple workflows. Here we introduce signal amplification by exchange reaction (SABER), which endows oligonucleotide-based FISH probes with long, single-stranded DNA concatemers that aggregate a multitude of short complementary fluorescent imager strands. We show that SABER amplified RNA and DNA FISH signals (5- to 450-fold) in fixed cells and tissues. We also applied 17 orthogonal amplifiers against chromosomal targets simultaneously and detected mRNAs with high efficiency. We then used 10-plex SABER-FISH to identify in vivo introduced enhancers with cell-type-specific activity in the mouse retina. SABER represents a simple and versatile molecular toolkit for rapid and cost-effective multiplexed imaging of nucleic acid targets.
2019-05-06Visualizing learner engagement, performance, and trajectories to evaluate and optimize online course designGinda M, Richey MC, Cousino M, Börner K.HIVE MC-IULearning analytics and visualizations make it possible to examine and communicate learners’ engagement, performance, and trajectories in online courses to evaluate and optimize course design for learners. This is particularly valuable for workforce training involving employees who need to acquire new knowledge in the most effective manner. This paper introduces a set of metrics and visualizations that aim to capture key dynamical aspects of learner engagement, performance, and course trajectories. The metrics are applied to identify prototypical behavior and learning pathways through and interactions with course content, activities, and assessments. The approach is exemplified and empirically validated using more than 30 million separate logged events that capture activities of 1,608 Boeing engineers taking the MITxPro Course, “Architecture of Complex Systems,” delivered in Fall 2016. Visualization results show course structure and patterns of learner interactions with course material, activities, and assessments. Tree visualizations are used to represent course hierarchical structures and explicit sequence of content modules. Learner trajectory networks represent pathways and interactions of individual learners through course modules, revealing patterns of learner engagement, content access strategies, and performance. Results provide evidence for instructors and course designers for evaluating the usage and effectiveness of course materials and intervention strategies.
2019-06-04Cell lineage inference from SNP and scRNA-Seq dataDing J, Lin C, Bar-Joseph Z.HIVE TC-CMUSeveral recent studies focus on the inference of developmental and response trajectories from single cell RNA-Seq (scRNA-Seq) data. A number of computational methods, often referred to as pseudo-time ordering, have been developed for this task. Recently, CRISPR has also been used to reconstruct lineage trees by inserting random mutations. However, both approaches suffer from drawbacks that limit their use. Here, we develop a method to detect significant, cell type specific, sequence mutations from scRNA-Seq data. We show that only a few mutations are enough for reconstructing good branching models. Integrating these mutations with expression data further improves the accuracy of the reconstructed models. As we show, the majority of mutations we identify are likely RNA editing events indicating that such information can be used to distinguish cell types.
2019-06-06Comprehensive Integration of Single-Cell DataStuart T, Butler A, Hoffman P, Hafemeister C, Papalexi E, Mauck WM 3rd, Hao Y, Stoeckius M, Smibert P, Satija R.HIVE MC-NYGCSingle-cell transcriptomics has transformed our ability to characterize cell states, but deep biological understanding requires more than a taxonomic listing of clusters. As new methods arise to measure distinct cellular modalities, a key analytical challenge is to integrate these datasets to better understand cellular identity and function. Here, we develop a strategy to “anchor” diverse datasets together, enabling us to integrate single-cell measurements not only across scRNA-seq technologies, but also across different modalities. After demonstrating improvement over existing methods for integrating scRNA-seq data, we anchor scRNA-seq experiments with scATAC-seq to explore chromatin differences in closely related interneuron subsets and project protein expression measurements onto a bone marrow atlas to characterize lymphocyte populations. Lastly, we harmonize in situ gene expression and scRNA-seq datasets, allowing transcriptome-wide imputation of spatial gene expression patterns. Our work presents a strategy for the assembly of harmonized references and transfer of information across datasets.
2019-06-07The human body at cellular resolution: the NIH Human Biomolecular Atlas ProgramHuBMAP ConsortiumConsortiumTransformative technologies are enabling the construction of three-dimensional maps of tissues with unprecedented spatial and molecular resolution. Over the next seven years, the NIH Common Fund Human Biomolecular Atlas Program (HuBMAP) intends to develop a widely accessible framework for comprehensively mapping the human body at single-cell resolution by supporting technology development, data acquisition, and detailed spatial mapping. HuBMAP will integrate its efforts with other funding agencies, programs, consortia, and the biomedical research community at large towards the shared vision of a comprehensive, accessible three-dimensional molecular and cellular atlas of the human body, in health and under various disease conditions.
2019-06-13Two Specific Sulfatide Species Are Dysregulated during Renal Development in a Mouse Model of Alport SyndromeGessel MM, Spraggins JM, Voziyan PA, Abrahamson DR, Caprioli RM, Hudson BG.TMC-Vanderbilt (Kidney)Alport syndrome is caused by mutations in collagen IV that alter the morphology of renal glomerular basement membrane. Mutations result in proteinuria, tubulointerstitial fibrosis, and renal failure but the pathogenic mechanisms are not fully understood. Using imaging mass spectrometry, we aimed to determine whether the spatial and/or temporal patterns of renal lipids are perturbed during the development of Alport syndrome in the mouse model. Our results show that most sulfatides are present at similar levels in both the wild-type (WT) and the Alport kidneys, with the exception of two specific sulfatide species, SulfoHex-Cer(d18:2/24:0) and SulfoHex-Cer(d18:2/16:0). In the Alport but not in WT kidneys, the levels of these species mirror the previously described abnormal laminin expression in Alport syndrome. The presence of these sulfatides in renal tubules but not in glomeruli suggests that this specific aberrant lipid pattern may be related to the development of tubulointerstitial fibrosis in Alport disease.
2019-06-18MicroLESA: Integrating Autofluorescence Microscopy, In Situ Micro-Digestions, and Liquid Extraction Surface Analysis for High Spatial Resolution Targeted Proteomic Studies.Ryan DJ, Patterson NH, Putnam NE, Wilde AD, Weiss A, Perry WJ, Cassat JE, Skaar EP, Caprioli RM, Spraggins JM.TMC-Vanderbilt (Kidney)The ability to target discrete features within tissue using liquid surface extractions enables the identification of proteins while maintaining the spatial integrity of the sample. Here, we present a liquid extraction surface analysis (LESA) workflow, termed microLESA, that allows proteomic profiling from discrete tissue features of ∼110 μm in diameter by integrating nondestructive autofluorescence microscopy and spatially targeted liquid droplet micro-digestion. Autofluorescence microscopy provides the visualization of tissue foci without the need for chemical stains or the use of serial tissue sections. Tryptic peptides are generated from tissue foci by applying small volume droplets (∼250 pL) of enzyme onto the surface prior to LESA. The microLESA workflow reduced the diameter of the sampled area almost 5-fold compared to previous LESA approaches. Experimental parameters, such as tissue thickness, trypsin concentration, and enzyme incubation duration, were tested to maximize proteomics analysis. The microLESA workflow was applied to the study of fluorescently labeled Staphylococcus aureus infected murine kidney to identify unique proteins related to host defense and bacterial pathogenesis. Proteins related to nutritional immunity and host immune response were identified by performing microLESA at the infectious foci and surrounding abscess. These identifications were then used to annotate specific proteins observed in infected kidney tissue by MALDI FT-ICR IMS through accurate mass matching.
2019-06-19The 2019 mathematical oncology roadmapRockne RC, Hawkins-Daarud A, Swanson KR, Sluka JP, Glazier JA, Macklin P, Hormuth DA, Jarrett AM, Lima EABF, Tinsley Oden J, Biros G, Yankeelov TE, Curtius K, Al Bakir I, Wodarz D, Komarova N, Aparicio L, Bordyuh M, Rabadan R, Finley SD, Enderling H, Caudell J, et al.HIVE MC-IUWhether the nom de guerre is Mathematical Oncology, Computational or Systems Biology, Theoretical Biology, Evolutionary Oncology, Bioinformatics, or simply Basic Science, there is no denying that mathematics continues to play an increasingly prominent role in cancer research. Mathematical Oncology—defined here simply as the use of mathematics in cancer research—complements and overlaps with a number of other fields that rely on mathematics as a core methodology. As a result, Mathematical Oncology has a broad scope, ranging from theoretical studies to clinical trials designed with mathematical models. This Roadmap differentiates Mathematical Oncology from related fields and demonstrates specific areas of focus within this unique field of research. The dominant theme of this Roadmap is the personalization of medicine through mathematics, modelling, and simulation. This is achieved through the use of patient-specific clinical data to: develop individualized screening strategies to detect cancer earlier; make predictions of response to therapy; design adaptive, patient-specific treatment plans to overcome therapy resistance; and establish domain-specific standards to share model predictions and to make models and simulations reproducible. The cover art for this Roadmap was chosen as an apt metaphor for the beautiful, strange, and evolving relationship between mathematics and cancer.
2019-08-01A recommended and verified procedure for in situ tryptic digestion of formalin-fixed paraffin-embedded tissues for analysis by matrix-assisted laser desorption/ionization imaging mass spectrometryJudd AM, Gutierrez DB, Moore JL, Patterson NH, Yang J, Romer CE, Norris JL, Caprioli RMTMC-Vanderbilt (Kidney)Matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI IMS) is a molecular imaging technology uniquely capable of untargeted measurement of proteins, lipids, and metabolites while retaining spatial information about their location in situ. This powerful combination of capabilities has the potential to bring a wealth of knowledge to the field of molecular histology. Translation of this innovative research tool into clinical laboratories requires the development of reliable sample preparation protocols for the analysis of proteins from formalin-fixed paraffin-embedded (FFPE) tissues, the standard preservation process in clinical pathology. Although ideal for stained tissue analysis by microscopy, the FFPE process cross-links, disrupts, or can remove proteins from the tissue, making analysis of the protein content challenging. To date, reported approaches differ widely in process and efficacy. This tutorial presents a strategy derived from systematic testing and optimization of key parameters, for reproducible in situ tryptic digestion of proteins in FFPE tissue and subsequent MALDI IMS analysis. The approach describes a generalized method for FFPE tissues originating from virtually any source.
2019-08-19Immuno-SABER enables highly multiplexed and amplified protein imaging in tissuesSaka SK, Wang Y, Kishi JY, Zhu A, Zeng Y, Xie W, Kirli K, Yapp C, Cicconet M, Beliveau BJ, Lapan SW, Yin S, Lin M, Boyden ES, Kaeser PS, Pihan G, Church GM, Yin P.TTD-HarvardSpatial mapping of proteins in tissues is hindered by limitations in multiplexing, sensitivity and throughput. Here we report immunostaining with signal amplification by exchange reaction (Immuno-SABER), which achieves highly multiplexed signal amplification via DNA-barcoded antibodies and orthogonal DNA concatemers generated by primer exchange reaction (PER). SABER offers independently programmable signal amplification without in situ enzymatic reactions, and intrinsic scalability to rapidly amplify and visualize a large number of targets when combined with fast exchange cycles of fluorescent imager strands. We demonstrate 5- to 180-fold signal amplification in diverse samples (cultured cells, cryosections, formalin-fixed paraffin-embedded sections and whole-mount tissues), as well as simultaneous signal amplification for ten different proteins using standard equipment and workflows. We also combined SABER with expansion microscopy to enable rapid, multiplexed super-resolution tissue imaging. Immuno-SABER presents an effective and accessible platform for multiplexed and amplified imaging of proteins with high sensitivity and throughput.
2019-09-02A pooled single-cell genetic screen identifies regulatory checkpoints in the continuum of the epithelial-to-mesenchymal transitionMcFaline-Figueroa JL, Hill AJ, Qiu X, Jackson D, Shendure J, Trapnell C.TMC-Cal TechIntegrating single-cell trajectory analysis with pooled genetic screening could reveal the genetic architecture that guides cellular decisions in development and disease. We applied this paradigm to probe the genetic circuitry that controls epithelial-to-mesenchymal transition (EMT). We used single-cell RNA sequencing to profile epithelial cells undergoing a spontaneous spatially determined EMT in the presence or absence of transforming growth factor-β. Pseudospatial trajectory analysis identified continuous waves of gene regulation as opposed to discrete ‘partial’ stages of EMT. KRAS was connected to the exit from the epithelial state and the acquisition of a fully mesenchymal phenotype. A pooled single-cell CRISPR-Cas9 screen identified EMT-associated receptors and transcription factors, including regulators of KRAS, whose loss impeded progress along the EMT. Inhibiting the KRAS effector MEK and its upstream activators EGFR and MET demonstrates that interruption of key signaling events reveals regulatory ‘checkpoints’ in the EMT continuum that mimic discrete stages, and reconciles opposing views of the program that controls EMT.
2019-09-10Supervised classification enables rapid annotation of cell atlasesPliner HA, Shendure J, Trapnell C.TMC-Cal TechSingle-cell molecular profiling technologies are gaining rapid traction, but the manual process by which resulting cell types are typically annotated is labor intensive and rate-limiting. We describe Garnett, a tool for rapidly annotating cell types in single-cell transcriptional profiling and single-cell chromatin accessibility datasets, based on an interpretable, hierarchical markup language of cell type-specific genes. Garnett successfully classifies cell types in tissue and whole organism datasets, as well as across species.
2019-10-07GiniClust3: a fast and memory-efficient tool for rare cell type identificationDong R, Yuan GC.TTD-Cal TechBACKGROUND: With the rapid development of single-cell RNA sequencing technology, it is possible to dissect cell-type composition at high resolution. A number of methods have been developed with the purpose to identify rare cell types. However, existing methods are still not scalable to large datasets, limiting their utility. To overcome this limitation, we present a new software package, called GiniClust3, which is an extension of GiniClust2 and significantly faster and memory-efficient than previous versions. RESULTS: Using GiniClust3, it only takes about 7 h to identify both common and rare cell clusters from a dataset that contains more than one million cells. Cell type mapping and perturbation analyses show that GiniClust3 could robustly identify cell clusters. CONCLUSIONS: Taken together, these results suggest that GiniClust3 is a powerful tool to identify both common and rare cell population and can handle large dataset. GiniCluster3 is implemented in the open-source python package and available at
2019-10-08High-Performance Molecular Imaging with MALDI Trapped Ion-Mobility Time-of-Flight (timsTOF) Mass SpectrometrySpraggins JM, Djambazova KV, Rivera ES, Migas LG, Neumann EK, Fuetterer A, Suetering J, Goedecke N, Ly A, Van de Plas R, Caprioli RM.TMC-Vanderbilt (Kidney)Understanding the genetic and molecular drivers of phenotypic heterogeneity across individuals is central to biology. As new technologies enable fine-grained and spatially resolved molecular profiling, we need new computational approaches to integrate data from the same organ across different individuals into a consistent reference and to construct maps of molecular and cellular organization at histological and anatomical scales. Here, we review previous efforts and discuss challenges involved in establishing such a common coordinate framework, the underlying map of tissues and organs. We focus on strategies to handle anatomical variation across individuals and highlight the need for new technologies and analytical methods spanning multiple hierarchical scales of spatial resolution.
2019-10-14High-throughput sequencing of the transcriptome and chromatin accessibility in the same cellChen S, Lake BB, Zhang K.TMC-UCSDSingle-cell RNA sequencing can reveal the transcriptional state of cells, yet provides little insight into the upstream regulatory landscape associated with open or accessible chromatin regions. Joint profiling of accessible chromatin and RNA within the same cells would permit direct matching of transcriptional regulation to its outputs. Here, we describe droplet-based single-nucleus chromatin accessibility and mRNA expression sequencing (SNARE-seq), a method that can link a cell’s transcriptome with its accessible chromatin for sequencing at scale. Specifically, accessible sites are captured by Tn5 transposase in permeabilized nuclei to permit, within many droplets in parallel, DNA barcode tagging together with the mRNA molecules from the same cells. To demonstrate the utility of SNARE-seq, we generated joint profiles of 5,081 and 10,309 cells from neonatal and adult mouse cerebral cortices, respectively. We reconstructed the transcriptome and epigenetic landscapes of major and rare cell types, uncovered lineage-specific accessible sites, especially for low-abundance cells, and connected the dynamics of promoter accessibility with transcription level during neurogenesis.
2019-11-13High spatial resolution imaging of biological tissues using nanospray desorption electrospray ionization mass spectrometryYin R, Burnum-Johnson KE, Sun X, Dey SK & Laskin JTTD-Purdue
2019-11-15Continuous State HMMs for Modeling Time Series Single Cell RNA-Seq DataLin C, Bar-Joseph Z.HIVE TC-CMUMOTIVATION: Methods for reconstructing developmental trajectories from time series single cell RNA-Seq (scRNA-Seq) data can be largely divided into two categories. The first, often referred to as pseudotime ordering methods, are deterministic and rely on dimensionality reduction followed by an ordering step. The second learns a probabilistic branching model to represent the developmental process. While both types have been successful, each suffers from shortcomings that can impact their accuracy. RESULTS: We developed a new method based on continuous state HMMs (CSHMMs) for representing and modeling time series scRNA-Seq data. We define the CSHMM model and provide efficient learning and inference algorithms which allow the method to determine both the structure of the branching process and the assignment of cells to these branches. Analyzing several developmental single cell datasets we show that the CSHMM method accurately infers branching topology and correctly and continuously assign cells to paths, improving upon prior methods proposed for this task. Analysis of genes based on the continuous cell assignment identifies known and novel markers for different cell types. AVAILABILITY: Software and Supporting website: SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.
2019-12-12Toward a Common Coordinate Framework for the Human BodyRood JE, Stuart T, Ghazanfar S, Biancalani T, Fisher E, Butler A, Hupalowska A, Gaffney L, Mauck W, Eraslan G, Marioni JC, Regev A, Satija R.HIVE MC-NYGCUnderstanding the genetic and molecular drivers of phenotypic heterogeneity across individuals is central to biology. As new technologies enable fine-grained and spatially resolved molecular profiling, we need new computational approaches to integrate data from the same organ across different individuals into a consistent reference and to construct maps of molecular and cellular organization at histological and anatomical scales. Here, we review previous efforts and discuss challenges involved in establishing such a common coordinate framework, the underlying map of tissues and organs. We focus on strategies to handle anatomical variation across individuals and highlight the need for new technologies and analytical methods spanning multiple hierarchical scales of spatial resolution.
2019-12-20Uncovering matrix effects on lipid analyses in MALDI imaging mass spectrometry experimentsPerry WJ, Patterson NH, Prentice BM, Neumann EK, Caprioli RM, Spraggins JM.TMC-Vanderbilt (Kidney)The specific matrix used in matrix‐assisted laser desorption/ionization imaging mass spectrometry (MALDI IMS) can have an effect on the molecules ionized from a tissue sample. The sensitivity for distinct classes of biomolecules can vary when employing different MALDI matrices. Here, we compare the intensities of various lipid subclasses measured by Fourier transform ion cyclotron resonance (FT‐ICR) IMS of murine liver tissue when using 9‐aminoacridine (9AA), 5‐chloro‐2‐mercaptobenzothiazole (CMBT), 1,5‐diaminonaphthalene (DAN), 2,5‐Dihydroxyacetophenone (DHA), and 2,5‐dihydroxybenzoic acid (DHB). Principal component analysis and receiver operating characteristic curve analysis revealed significant matrix effects on the relative signal intensities observed for different lipid subclasses and adducts. Comparison of spectral profiles and quantitative assessment of the number and intensity of species from each lipid subclass showed that each matrix produces unique lipid signals. In positive ion mode, matrix application methods played a role in the MALDI analysis for different cationic species. Comparisons of different methods for the application of DHA showed a significant increase in the intensity of sodiated and potassiated analytes when using an aerosol sprayer. In negative ion mode, lipid profiles generated using DAN were significantly different than all other matrices tested. This difference was found to be driven by modification of phosphatidylcholines during ionization that enables them to be detected in negative ion mode. These modified phosphatidylcholines are isomeric with common phosphatidylethanolamines confounding MALDI IMS analysis when using DAN. These results show an experimental basis of MALDI analyses when analyzing lipids from tissue and allow for more informed selection of MALDI matrices when performing lipid IMS experiments.
2019-12-23Normalization and variance stabilization of single-cell RNA-seq data using regularized negative binomial regressionHafemeister C, Satija R.HIVE MC-NYGCSingle-cell RNA-seq (scRNA-seq) data exhibits significant cell-to-cell variation due to technical factors, including the number of molecules detected in each cell, which can confound biological heterogeneity with technical effects. To address this, we present a modeling framework for the normalization and variance stabilization of molecular count data from scRNA-seq experiments. We propose that the Pearson residuals from “regularized negative binomial regression,” where cellular sequencing depth is utilized as a covariate in a generalized linear model, successfully remove the influence of technical characteristics from downstream analyses while preserving biological heterogeneity. Importantly, we show that an unconstrained negative binomial model may overfit scRNA-seq data, and overcome this by pooling information across genes with similar abundances to obtain stable parameter estimates. Our procedure omits the need for heuristic steps including pseudocount addition or log-transformation and improves common downstream analytical tasks such as variable gene selection, dimensional reduction, and differential expression. Our approach can be applied to any UMI-based scRNA-seq dataset and is freely available as part of the R package sctransform, with a direct interface to our single-cell toolkit Seurat.
2019-12-26Deep learning for inferring gene relationships from single-cell expression dataYuan Y, Bar-Joseph Z.HIVE TC-CMUSeveral methods were developed to mine gene–gene relationships from expression data. Examples include correlation and mutual information methods for coexpression analysis, clustering and undirected graphical models for functional assignments, and directed graphical models for pathway reconstruction. Using an encoding for gene expression data, followed by deep neural networks analysis, we present a framework that can successfully address all of these diverse tasks. We show that our method, convolutional neural network for coexpression (CNNC), improves upon prior methods in tasks ranging from predicting transcription factor targets to identifying disease-related genes to causality inference. CNNC’s encoding provides insights about some of the decisions it makes and their biological basis. CNNC is flexible and can easily be extended to integrate additional types of genomics data, leading to further improvements in its performance.
2019-12-31Immune monitoring using mass cytometry and related high-dimensional imaging approachesHartmann FJ, Bendall SC.RTI-StanfordThe cellular complexity and functional diversity of the human immune system necessitate the use of high-dimensional single-cell tools to uncover its role in multifaceted diseases such as rheumatic diseases, as well as other autoimmune and inflammatory disorders. Proteomic technologies that use elemental (heavy metal) reporter ions, such as mass cytometry (also known as CyTOF) and analogous high-dimensional imaging approaches (including multiplexed ion beam imaging (MIBI) and imaging mass cytometry (IMC)), have been developed from their low-dimensional counterparts, flow cytometry and immunohistochemistry, to meet this need. A growing number of studies have been published that use these technologies to identify functional biomarkers and therapeutic targets in rheumatic diseases, but the full potential of their application to rheumatic disease research has yet to be fulfilled. This Review introduces the underlying technologies for high-dimensional immune monitoring and discusses aspects necessary for their successful implementation, including study design principles, analytical tools and future developments for the field of rheumatology.
2020-01-07Automated mass spectrometry imaging of over 2000 proteins from tissue sections at 100-μm spatial resolutionPiehowski PD, Zhu Y, Bramer LM, Stratton KG, Zhao R, Orton DJ, Moore RJ, Yuan J, Mitchell HD, Gao Y, Webb-Robertson BM, Dey SK, Kelly RT, Burnum-Johnson KE.TTD-Purdue
2020-02-18Inferring TF activation order in time series scRNA-Seq studiesLin C, Ding J, Bar-Joseph Z.HIVE TC-CMUMethods for the analysis of time series single cell expression data (scRNA-Seq) either do not utilize information about transcription factors (TFs) and their targets or only study these as a post-processing step. Using such information can both, improve the accuracy of the reconstructed model and cell assignments, while at the same time provide information on how and when the process is regulated. We developed the Continuous-State Hidden Markov Models TF (CSHMM-TF) method which integrates probabilistic modeling of scRNA-Seq data with the ability to assign TFs to specific activation points in the model. TFs are assumed to influence the emission probabilities for cells assigned to later time points allowing us to identify not just the TFs controlling each path but also their order of activation. We tested CSHMM-TF on several mouse and human datasets. As we show, the method was able to identify known and novel TFs for all processes, assigned time of activation agrees with both expression information and prior knowledge and combinatorial predictions are supported by known interactions. We also show that CSHMM-TF improves upon prior methods that do not utilize TF-gene interaction
2020-03-11Multiplexed single-cell morphometry for hematopathology diagnosticsTsai AG, Glass DR, Juntilla M, Hartmann FJ, Oak JS, Fernandez-Pol S, Ohgami RS, Bendall SC.RTI-StanfordThe diagnosis of lymphomas and leukemias requires hematopathologists to integrate microscopically visible cellular morphology with antibody-identified cell surface molecule expression. To merge these into one high-throughput, highly multiplexed, single-cell assay, we quantify cell morphological features by their underlying, antibody-measurable molecular components, which empowers mass cytometers to ‘see’ like pathologists. When applied to 71 diverse clinical samples, single-cell morphometric profiling reveals robust and distinct patterns of ‘morphometric’ markers for each major cell type. Individually, lamin B1 highlights acute leukemias, lamin A/C helps distinguish normal from neoplastic mature T cells, and VAMP-7 recapitulates light-cytometric side scatter. Combined with machine learning, morphometric markers form intuitive visualizations of normal and neoplastic cellular distribution and differentiation. When recalibrated for myelomonocytic blast enumeration, this approach is superior to flow cytometry and comparable to expert microscopy, bypassing years of specialized training. The contextualization of traditional surface markers on independent morphometric frameworks permits more sensitive and automated diagnosis of complex hematopoietic diseases.
2020-03-13Considerations for Using the Vasculature as a Coordinate System to Map All the Cells in the Human BodyWeber, GM, Ju, Y, Börner K.HIVE MC-IUSeveral ongoing international efforts are developing methods of localizing single cells within organs or mapping the entire human body at the single cell level, including the Chan Zuckerberg Initiative’s Human Cell Atlas (HCA), and the Knut and Allice Wallenberg Foundation’s Human Protein Atlas (HPA), and the National Institutes of Health’s Human BioMolecular Atlas Program (HuBMAP). Their goals are to understand cell specialization, interactions, spatial organization in their natural context, and ultimately the function of every cell within the body. In the same way that the Human Genome Project had to assemble sequence data from different people to construct a complete sequence, multiple centers around the world are collecting tissue specimens from diverse populations that vary in age, race, sex, and body size. A challenge will be combining these heterogeneous tissue samples into a 3D reference map that will enable multiscale, multidimensional Google Maps-like exploration of the human body. Key to making alignment of tissue samples work is identifying and using a coordinate system called a Common Coordinate Framework (CCF), which defines the positions, or “addresses,” in a reference body, from whole organs down to functional tissue units and individual cells. In this perspective, we examine the concept of a CCF based on the vasculature and describe why it would be an attractive choice for mapping the human body.
2020-03-27Tools for the analysis of high-dimensional single-cell RNA sequencing dataWu Y, Zhang K.TMC-UCSDBreakthroughs in the development of high-throughput technologies for profiling transcriptomes at the single-cell level have helped biologists to understand the heterogeneity of cell populations, disease states and developmental lineages. However, these single-cell RNA sequencing (scRNA-seq) technologies generate an extraordinary amount of data, which creates analysis and interpretation challenges. Additionally, scRNA-seq datasets often contain technical sources of noise owing to incomplete RNA capture, PCR amplification biases and/or batch effects specific to the patient or sample. If not addressed, this technical noise can bias the analysis and interpretation of the data. In response to these challenges, a suite of computational tools has been developed to process, analyse and visualize scRNA-seq datasets. Although the specific steps of any given scRNA-seq analysis might differ depending on the biological questions being asked, a core workflow is used in most analyses. Typically, raw sequencing reads are processed into a gene expression matrix that is then normalized and scaled to remove technical noise. Next, cells are grouped according to similarities in their patterns of gene expression, which can be summarized in two or three dimensions for visualization on a scatterplot. These data can then be further analysed to provide an in-depth view of the cell types or developmental trajectories in the sample of interest.
2020-04-01Integrated molecular imaging technologies for investigation of metals in biological systems: A brief reviewPerry WJ, Weiss A, Van de Plas R, Spraggins JM, Caprioli RM, Skaar EP.TMC-Vanderbilt (Kidney)Metals play an essential role in biological systems and are required as structural or catalytic co-factors in many proteins. Disruption of the homeostatic control and/or spatial distributions of metals can lead to disease. Imaging technologies have been developed to visualize elemental distributions across a biological sample. Measurement of elemental distributions by imaging mass spectrometry and imaging X-ray fluorescence are increasingly employed with technologies that can assess histological features and molecular compositions. Data from several modalities can be interrogated as multimodal images to correlate morphological, elemental, and molecular properties. Elemental and molecular distributions have also been axially resolved to achieve three-dimensional volumes, dramatically increasing the biological information. In this review, we provide an overview of recent developments in the field of metal imaging with an emphasis on multimodal studies in two and three dimensions. We specifically highlight studies that present technological advancements and biological applications of how metal homeostasis affects human health.
2020-04-01RETrace: simultaneous retrospective lineage tracing and methylation profiling of single cellsWei CJ, Zhang K.Retrospective lineage tracing harnesses naturally occurring mutations in cells to elucidate single cell development. Common single-cell phylogenetic fate mapping methods have utilized highly mutable microsatellite loci found within the human genome. Such methods were limited by the introduction of in vitro noise through polymerase slippage inherent in DNA amplification, which we characterized to be approximately 10–100× higher than the in vivo replication mutation rate. Here, we present RETrace, a method for simultaneously capturing both microsatellites and methylation-informative cytosines to characterize both lineage and cell type, respectively, from the same single cell. An important unique feature of RETrace was the introduction of linear amplification of microsatellites in order to reduce in vitro amplification noise. We further coupled microsatellite capture with single-cell reduced representation bisulfite sequencing (scRRBS), to measure the CpG methylation status on the same cell for cell type inference. When compared to existing retrospective lineage tracing methods, RETrace achieved higher accuracy (88% triplet accuracy from an ex vivo HCT116 tree) at a higher cell division resolution (lowering the required number of cell division difference between single cells by approximately 100 divisions). Simultaneously, RETrace demonstrated the ability to capture on average 150,000 unique CpGs per single cell in order to accurately determine cell type. We further formulated additional developments that would allow high-resolution mapping on microsatellite-stable cells or tissues with RETrace. Overall, we present RETrace as a foundation for multi-omics lineage mapping and cell typing of single cells.
2020-04-02Reconstructed Single-Cell Fate Trajectories Define Lineage Plasticity Windows during Differentiation of Human PSC-Derived Distal Lung ProgenitorsHurley K, Ding J, Villacorta-Martin C, Herriges MJ, Jacob A, Vedaie M, Alysandratos KD, Sun YL, Lin C, Werder RB, Huang J, Wilson AA, Mithal A, Mostoslavsky G, Oglesby I, Caballero IS, Guttentag SH, Ahangari F, Kaminski N, Rodriguez-Fraticelli A, Camargo F, Bar-Joseph Z, Kotton DN.HIVE TC-CMUAlveolar epithelial type 2 cells (AEC2s) are the facultative progenitors responsible for maintaining lung alveoli throughout life but are difficult to isolate from patients. Here, we engineer AEC2s from human pluripotent stem cells (PSCs) in vitro and use time-series single-cell RNA sequencing with lentiviral barcoding to profile the kinetics of their differentiation in comparison to primary fetal and adult AEC2 benchmarks. We observe bifurcating cell-fate trajectories as primordial lung progenitors differentiate in vitro, with some progeny reaching their AEC2 fate target, while others diverge to alternative non-lung endodermal fates. We develop a Continuous State Hidden Markov model to identify the timing and type of signals, such as overexuberant Wnt responses, that induce some early multipotent NKX2-1+ progenitors to lose lung fate. Finally, we find that this initial developmental plasticity is regulatable and subsides over time, ultimately resulting in PSC-derived AEC2s that exhibit a stable phenotype and nearly limitless self-renewal capacity.
2020-05-01Unsupervised machine learning for exploratory data analysis in imaging mass spectrometry. Screen reader support enabledVerbeeck N, Caprioli RM, Van de Plas R.TMC-Vanderbilt (Kidney)Imaging mass spectrometry (IMS) is a rapidly advancing molecular imaging modality that can map the spatial distribution of molecules with high chemical specificity. IMS does not require prior tagging of molecular targets and is able to measure a large number of ions concurrently in a single experiment. While this makes it particularly suited for exploratory analysis, the large amount and high‐dimensional nature of data generated by IMS techniques make automated computational analysis indispensable. Research into computational methods for IMS data has touched upon different aspects, including spectral preprocessing, data formats, dimensionality reduction, spatial registration, sample classification, differential analysis between IMS experiments, and data‐driven fusion methods to extract patterns corroborated by both IMS and other imaging modalities. In this work, we review unsupervised machine learning methods for exploratory analysis of IMS data, with particular focus on (a) factorization, (b) clustering, and (c) manifold learning. To provide a view across the various IMS modalities, we have attempted to include examples from a range of approaches including matrix assisted laser desorption/ionization, desorption electrospray ionization, and secondary ion mass spectrometry‐based IMS. This review aims to be an entry point for both (i) analytical chemists and mass spectrometry experts who want to explore computational techniques; and (ii) computer scientists and data mining specialists who want to enter the IMS field.
2020-05-14Use of Single Cell -omic Technologies to Study the Gastrointestinal Tract and Diseases, From Single Cell Identities to Patient FeaturesIslam M, Chen B, Spraggins JM, Kelly RT, Lau KS.TMC-Vanderbilt (Kidney)Single cells are the building blocks of tissue systems that determine organ phenotypes, behaviors, and function. Understanding the differences between cell types and their activities might provide us with insights into normal tissue functions, development of disease, and new therapeutic strategies. Although -omic level single cell technologies are a relatively recent development that been used only in laboratory studies, these approaches might eventually be used in the clinic. We review the prospects of applying single cell genome, transcriptome, epigenome, proteome, and metabolome analyses to gastroenterology and hepatology research. Combining data from multi-omic platforms and rapid technological developments could lead to new diagnostic, prognostic, and therapeutic approaches.
2020-05-19Discovering New Lipidomic Features Using Cell Type Specific Fluorophore Expression to Provide Spatial and Biological Specificity in a Multimodal Workflow with MALDI Imaging Mass SpectrometryJones MA, Cho SH, Patterson NH, Van de Plas R, Spraggins JM, Boothby MR, Caprioli RM.TMC-Vanderbilt (Kidney)Identifying the spatial distributions of biomolecules in tissue is crucial for understanding integrated function. Imaging mass spectrometry (IMS) allows simultaneous mapping of thousands of biosynthetic products such as lipids but has needed a means of identifying specific cell-types or functional states to correlate with molecular localization. We report, here, advances starting from identity marking with a genetically encoded fluorophore. The fluorescence emission data were integrated with IMS data through multimodal image processing with advanced registration techniques and data-driven image fusion. In an unbiased analysis of spleens, this integrated technology enabled identification of ether lipid species preferentially enriched in germinal centers. We propose that this use of genetic marking for microanatomical regions of interest can be paired with molecular information from IMS for any tissue, cell-type, or activity state for which fluorescence is driven by a gene-tracking allele and ultimately with outputs of other means of spatial mapping.
2020-06-16Single-cell Lineage Tracing by Integrating CRISPR-Cas9 Mutations With Transcriptomic DataZafar H, Lin C, Bar-Joseph Z.HIVE TC-CMURecent studies combine two novel technologies, single-cell RNA-sequencing and CRISPR-Cas9 barcode editing for elucidating developmental lineages at the whole organism level. While these studies provided several insights, they face several computational challenges. First, lineages are reconstructed based on noisy and often saturated random mutation data. Additionally, due to the randomness of the mutations, lineages from multiple experiments cannot be combined to reconstruct a species-invariant lineage tree. To address these issues we developed a statistical method, LinTIMaT, which reconstructs cell lineages using a maximum-likelihood framework by integrating mutation and expression data. Our analysis shows that expression data helps resolve the ambiguities arising in when lineages are inferred based on mutations alone, while also enabling the integration of different individual lineages for the reconstruction of an invariant lineage tree. LinTIMaT lineages have better cell type coherence, improve the functional significance of gene sets and provide new insights on progenitors and differentiation pathways.
2020-06-30A Cancer Biologist's Primer on Machine Learning Applications in High-Dimensional CytometryKeyes TJ, Domizi P, Lo YC, Nolan GP, Davis KLTMC-StanfordThe application of machine learning and artificial intelligence to high-dimensional cytometry data sets has increasingly become a staple of bioinformatic data analysis over the past decade. This is especially true in the field of cancer biology, where protocols for collecting multiparameter single-cell data in a high-throughput fashion are rapidly developed. As the use of machine learning methodology in cytometry becomes increasingly common, there is a need for cancer biologists to understand the basic theory and applications of a variety of algorithmic tools for analyzing and interpreting cytometry data. We introduce the reader to several keystone machine learning-based analytic approaches with an emphasis on defining key terms and introducing a conceptual framework for making translational or clinically relevant discoveries. The target audience consists of cancer cell biologists and physician-scientists interested in applying these tools to their own data, but who may have limited training in bioinformatics. © 2020 International Society for Advancement of Cytometry.
2020-07-14An Integrated Multi-omic Single-Cell Atlas of Human B Cell Identity.Glass DR, Tsai AG, Oliveria JP, Hartmann FJ, Kimmey SC, Calderon AA, Borges L, Glass MC, Wagar LE, Davis MM, Bendall SC.RTI-StanfordB cells are capable of a wide range of effector functions including antibody secretion, antigen presentation, cytokine production, and generation of immunological memory. A consistent strategy for classifying human B cells by using surface molecules is essential to harness this functional diversity for clinical translation. We developed a highly multiplexed screen to quantify the co-expression of 351 surface molecules on millions of human B cells. We identified differentially expressed molecules and aligned their variance with isotype usage, VDJ sequence, metabolic profile, biosynthesis activity, and signaling response. Based on these analyses, we propose a classification scheme to segregate B cells from four lymphoid tissues into twelve unique subsets, including a CD45RB+CD27- early memory population, a class-switched CD39+ tonsil-resident population, and a CD19hiCD11c+ memory population that potently responds to immune activation. This classification framework and underlying datasets provide a resource for further investigations of human B cell identity and function.
2020-07-23Multimodal Analysis of Composition and Spatial Architecture in Human Squamous Cell CarcinomaJi AL, Rubin AJ, Thrane K, Jiang S, Reynolds DL, Meyers RM, Guo MG, George BM, Mollbrink A, Bergenstråhle J, Larsson L, Bai Y, Zhu B, Bhaduri A, Meyers JM, Rovira-Clavé X, Hollmig ST, Aasi SZ, Nolan GP, Lundeberg J, Khavari PATMC-StanfordTo define the cellular composition and architecture of cutaneous squamous cell carcinoma (cSCC), we combined single-cell RNA sequencing with spatial transcriptomics and multiplexed ion beam imaging from a series of human cSCCs and matched normal skin. cSCC exhibited four tumor subpopulations, three recapitulating normal epidermal states, and a tumor-specific keratinocyte (TSK) population unique to cancer, which localized to a fibrovascular niche. Integration of single-cell and spatial data mapped ligand-receptor networks to specific cell types, revealing TSK cells as a hub for intercellular communication. Multiple features of potential immunosuppression were observed, including T regulatory cell (Treg) co-localization with CD8 T cells in compartmentalized tumor stroma. Finally, single-cell characterization of human tumor xenografts and in vivo CRISPR screens identified essential roles for specific tumor subpopulation-enriched gene networks in tumorigenesis. These data define cSCC tumor and stromal cell subpopulations, the spatial niches where they interact, and the communicating gene networks that they engage in cancer.
2020-08-31Single-cell metabolic profiling of human cytotoxic T cellsHartmann FJ, Mrdjen D, McCaffrey E, Glass DR, Greenwald NF, Bharadwaj A, Khair Z, Verberk SGS, Baranski A, Baskar R, Graf W, Van Valen D, Van den Bossche J, Angelo M, Bendall SC.RTI-StanfordCellular metabolism regulates immune cell activation, differentiation and effector functions, but current metabolic approaches lack single-cell resolution and simultaneous characterization of cellular phenotype. In this study, we developed an approach to characterize the metabolic regulome of single cells together with their phenotypic identity. The method, termed single-cell metabolic regulome profiling (scMEP), quantifies proteins that regulate metabolic pathway activity using high-dimensional antibody-based technologies. We employed mass cytometry (cytometry by time of flight, CyTOF) to benchmark scMEP against bulk metabolic assays by reconstructing the metabolic remodeling of in vitro-activated naive and memory CD8+ T cells. We applied the approach to clinical samples and identified tissue-restricted, metabolically repressed cytotoxic T cells in human colorectal carcinoma. Combining our method with multiplexed ion beam imaging by time of flight (MIBI-TOF), we uncovered the spatial organization of metabolic programs in human tissues, which indicated exclusion of metabolically repressed immune cells from the tumor-immune boundary. Overall, our approach enables robust approximation of metabolic and functional states in individual cells.
2020-09-01Nanopore sequencing and the Shasta toolkit enable efficient de novo assembly of eleven human genomesShafin K, Pesout T, Lorig-Roach R, Haukness M, Olsen HE, Bosworth C, Armstrong J, Tigyi K, Maurer N, Koren S, Sedlazeck FJ, Marschall T, Mayes S, Costa V, Zook JM, Liu KJ, Kilburn D, Sorensen M, Munson KM, Vollger MR, Monlong J, Garrison E, Eichler EE, Salama S, Haussler D, Green RE, Akeson M, Phillippy A, Miga KH, Carnevali P, Jain M, Paten BHIVE TC-CMUDe novo assembly of a human genome using nanopore long-read sequences has been reported, but it used more than 150,000 CPU hours and weeks of wall-clock time. To enable rapid human genome assembly, we present Shasta, a de novo long-read assembler, and polishing algorithms named MarginPolish and HELEN. Using a single PromethION nanopore sequencer and our toolkit, we assembled 11 highly contiguous human genomes de novo in 9 d. We achieved roughly 63× coverage, 42-kb read N50 values and 6.5× coverage in reads >100 kb using three flow cells per sample. Shasta produced a complete haploid human genome assembly in under 6 h on a single commercial compute node. MarginPolish and HELEN polished haploid assemblies to more than 99.9% identity (Phred quality score QV = 30) with nanopore reads alone. Addition of proximity-ligation sequencing enabled near chromosome-level scaffolds for all 11 genomes. We compare our assembly performance to existing methods for diploid, haploid and trio-binned human samples and report superior accuracy and speed.
2020-09-03Coordinated Cellular Neighborhoods Orchestrate Antitumoral Immunity at the Colorectal Cancer Invasive FrontSchürch CM, Bhate SS, Barlow GL, Phillips DJ, Noti L, Zlobec I, Chu P, Black S, Demeter J, McIlwain DR, Kinoshita S, Samusik N, Goltsev Y, Nolan GP.TMC-StanfordAntitumoral immunity requires organized, spatially nuanced interactions between components of the immune tumor microenvironment (iTME). Understanding this coordinated behavior in effective versus ineffective tumor control will advance immunotherapies. We re-engineered co-detection by indexing (CODEX) for paraffin-embedded tissue microarrays, enabling simultaneous profiling of 140 tissue regions from 35 advanced-stage colorectal cancer (CRC) patients with 56 protein markers. We identified nine conserved, distinct cellular neighborhoods (CNs)-a collection of components characteristic of the CRC iTME. Enrichment of PD-1+CD4+ T cells only within a granulocyte CN positively correlated with survival in a high-risk patient subset. Coupling of tumor and immune CNs, fragmentation of T cell and macrophage CNs, and disruption of inter-CN communication was associated with inferior outcomes. This study provides a framework for interrogating how complex biological processes, such as antitumoral immunity, occur through concerted actions of cells and spatial domains.
2020-09-04The impact of air transport availability on research collaboration: A case study of four universitiesPloszaj A, Yan X, Börner K.HIVE MC-IUThis paper analyzes the impact of air transport connectivity and accessibility on scientific collaboration. Numerous studies demonstrated that the likelihood of collaboration declines with increase in distance between potential collaborators. These works commonly use simple measures of physical distance rather than actual flight capacity and frequency. Our study addresses this limitation by focusing on the relationship between flight availability and the number of scientific co-publications. Furthermore, we distinguish two components of flight availability: (1) direct and indirect air connections between airports; and (2) distance to the nearest airport from cities and towns where authors of scientific articles have their professional affiliations. Based on Zero-inflated Negative Binomial Regression, we provide evidence that greater flight availability is associated with more frequent scientific collaboration. More flight connections (connectivity) and proximity of airport (accessibility) increase the expected number of coauthored scientific papers. Moreover, direct flights and flights with one transfer are more valuable for intensifying scientific cooperation than travels involving more connecting flights. Further, analysis of four organizational sub-datasets-Arizona State University, Indiana University Bloomington, Indiana University-Purdue University Indianapolis, and University of Michigan-shows that the relationship between airline transport availability and scientific collaboration is not uniform, but is associated with the research profile of an institution and the characteristics of the airport that serves this institution.
2020-09-29Targeting Phosphotyrosine in Native Proteins with Conditional, Bispecific Antibody TrapsZhou XX, Bracken CJ, Zhang K, Zhou J, Mou Y, Wang L, Cheng Y, Leung KK, Wells JA.RTI-NorthwesternEngineering sequence-specific antibodies (Abs) against phosphotyrosine (pY) motifs embedded in folded polypeptides remains highly challenging because of the stringent requirement for simultaneous recognition of the pY motif and the surrounding folded protein epitope. Here, we present a method named phosphotyrosine Targeting by Recombinant Ab Pair, or pY-TRAP, for in vitro engineering of binders for native pY proteins. Specifically, we create the pY protein by unnatural amino acid misincorporation, mutagenize a universal pY-binding Ab to create a first binder B1 for the pY motif on the pY protein, and then select against the B1-pY protein complex for a second binder B2 that recognizes the composite epitope of B1 and the pY-containing protein complex. We applied pY-TRAP to create highly specific binders to folded Ub-pY59, a rarely studied Ub phosphoform exclusively observed in cancerous tissues, and ZAP70-pY248, a kinase phosphoform regulated in feedback signaling pathways in T cells. The pY-TRAPs do not have detectable binding to wild-type proteins or to other pY peptides or proteins tested. This pY-TRAP approach serves as a generalizable method for engineering sequence-specific Ab binders to native pY proteins.
2020-10-09An Integrated Microfluidic Probe for Mass Spectrometry Imaging of Biological Samples*Li X, Yin R, Hu H, Li Y, Sun X, Dey SK, Laskin J.TTD-PurdueAmbient ionization based on liquid extraction is widely used in mass spectrometry imaging (MSI) of molecules in biological samples. The development of nanospray desorption electrospray ionization (nano-DESI) has enabled the robust imaging of tissue sections with high spatial resolution. However, the fabrication of the nano-DESI probe is challenging, which limits its dissemination to the broader scientific community. Herein, we describe the design and performance of an integrated microfluidic probe (iMFP) for nano-DESI MSI. The glass iMFP, fabricated using photolithography, wet etching, and polishing, shows comparable performance to the capillary-based nano-DESI MSI in terms of stability and sensitivity; a spatial resolution of better than 25 μm was obtained in these first proof-of-principle experiments. The iMFP is easy to operate and align in front of a mass spectrometer, which will facilitate broader use of liquid-extraction-based MSI in biological research, drug discovery, and clinical studies.
2020-10-19CDKL5: a promising new therapeutic target for acute kidney injury?de Caestecker MP.TMC-Vanderbilt (Kidney)Online ahead of print. No abstract available.
2020-10-27Iterative point set registration for aligning scRNA-seq dataAlavi A, Bar-Joseph ZHIVE TC-CMUSeveral studies profile similar single cell RNA-Seq (scRNA-Seq) data using different technologies and platforms. A number of alignment methods have been developed to enable the integration and comparison of scRNA-Seq data from such studies. While each performs well on some of the datasets, to date no method was able to both perform the alignment using the original expression space and generalize to new data. To enable such analysis we developed Single Cell Iterative Point set Registration (SCIPR) which extends methods that were successfully applied to align image data to scRNA-Seq. We discuss the required changes needed, the resulting optimization function, and algorithms for learning a transformation function for aligning data. We tested SCIPR on several scRNA-Seq datasets. As we show it successfully aligns data from several different cell types, improving upon prior methods proposed for this task. In addition, we show the parameters learned by SCIPR can be used to align data not used in the training and to identify key cell type-specific genes.
2020-11-01High-Parameter Immune Profiling with CyTOFSahaf B, Rahman A, Maecker HT, Bendall SCRTI-StanfordMass cytometry, or CyTOF, is a useful technology for high-parameter single-cell phenotyping, especially from suspension cells such as blood or PBMC. It is particularly appealing to monitor the systemic immune changes that could accompany cancer immunotherapy. Here we present a reference panel for identification of all major immune cell populations, with flexibility for addition of trial-specific markers. We also describe best-practice measures for minimizing and tracking batch variability. These include: sample barcoding, use of spiked-in reference cells, and lyophilization of the antibody cocktail. Our protocol assumes the use of cryopreserved PBMC, both for convenience of batching samples and for maximum comparability across patients and time points. Finally, we show an option for automated analysis using the Astrolabe platform (Astrolabe Diagnostics, Inc.).
2020-11-02Landscape of coordinated immune responses to H1N1 challenge in humans.Rahil Z, Leylek R, Schürch CM, Chen H, Bjornson-Hooper Z, Christensen SR, Gherardini PF, Bhate SS, Spitzer MH, Fragiadakis GK, Mukherjee N, Kim N, Jiang S, Yo J, Gaudilliere B, Affrime M, Bock B, Hensley SE, Idoyaga J, Aghaeepour N, Kim K, Nolan GP, McIlwain DR.TMC-StanfordInfluenza is a significant cause of morbidity and mortality worldwide. Here we show changes in the abundance and activation states of more than 50 immune cell subsets in 35 individuals over 11 time points during human A/California/2009 (H1N1) virus challenge monitored using mass cytometry along with other clinical assessments. Peak change in monocyte, B cell, and T cell subset frequencies coincided with peak virus shedding, followed by marked activation of T and NK cells. Results led to the identification of CD38 as a critical regulator of plasmacytoid dendritic cell function in response to influenza virus. Machine learning using study-derived clinical parameters and single-cell data effectively classified and predicted susceptibility to infection. The coordinated immune cell dynamics defined in this study provide a framework for identifying novel correlates of protection in the evaluation of future influenza therapeutics.
2020-11-05Advances in Proximity Ligation in situ Hybridization (PLISH)Nagendran M, Andruska AM, Harbury PB, Desai TJ.TTD-StanfordUnderstanding tissues in the context of development, maintenance and disease requires determining the molecular profiles of individual cells within their native in vivo spatial context. We developed a Proximity Ligation in situ Hybridization technology (PLISH) that enables quantitative measurement of single cell gene expression in intact tissues, which we have now updated. By recording spatial information for every profiled cell, PLISH enables retrospective mapping of distinct cell classes and inference of their in vivo interactions. PLISH has high sensitivity, specificity and signal to noise ratio. It is also rapid, scalable, and does not require expertise in molecular biology so it can be easily adopted by basic and clinical researchers.
2020-11-06Carrier-assisted One-pot Sample Preparation for Targeted Proteomics Analysis of Small Numbers of Human CellsMartin K, Zhang T, Zhang P, Chrisler WB, Thomas FL, Liu F, Liu T, Qian WJ, Smith RD, Shi T.TMC-PNNLProtein analysis of small numbers of human cells is primarily achieved by targeted proteomics with antibody-based immunoassays, which have inherent limitations (e.g., low multiplex and unavailability of antibodies for new proteins). Mass spectrometry (MS)-based targeted proteomics has emerged as an alternative because it is antibody-free, high multiplex, and has high specificity and quantitation accuracy. Recent advances in MS instrumentation make MS-based targeted proteomics possible for multiplexed quantification of highly abundant proteins in single cells. However, there is a technical challenge for effective processing of single cells with minimal sample loss for MS analysis. To address this issue, we have recently developed a convenient protein carrier-assisted one-pot sample preparation coupled with liquid chromatography (LC) - selected reaction monitoring (SRM) termed cLC-SRM for targeted proteomics analysis of small numbers of human cells. This method capitalizes on using the combined excessive exogenous protein as a carrier and low-volume one-pot processing to greatly reduce surface adsorption losses as well as high-specificity LC-SRM to effectively address the increased dynamic concentration range due to the addition of exogeneous carrier protein. Its utility has been demonstrated by accurate quantification of most moderately abundant proteins in small numbers of cells (e.g., 10-100 cells) and highly abundant proteins in single cells. The easy-to-implement features and no need for specific devices make this method readily accessible to most proteomics laboratories. Herein we have provided a detailed protocol for cLC-SRM analysis of small numbers of human cells including cell sorting, cell lysis and digestion, LC-SRM analysis, and data analysis. Further improvements in detection sensitivity and sample throughput are needed towards targeted single-cell proteomics analysis. We anticipate that cLC-SRM will be broadly applied to biomedical research and systems biology with the potential of facilitating precision medicine.
2020-11-13Guidelines for reporting single-cell RNA-seq experiments.Füllgrabe A, George N, Green M, Nejad P, Aronow B, Fexova SK, Fischer C, Freeberg MA, Huerta L, Morrison N, Scheuermann RH, Taylor D, Vasilevsky N, Clarke L, Gehlenborg N, Kent J, Marioni J, Teichmann S, Brazma A, Papatheodorou IHIVE TC-HarvardNo abstract available.
2020-12-01Effect of MALDI matrices on lipid analyses of biological tissues using MALDI-2 postionization mass spectrometryMcMillen JC, Fincher JA, Klein DR, Spraggins JM, Caprioli RMTMC-Vanderbilt (Kidney)Matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI IMS) allows for highly multiplexed, untargeted detection of many hundreds of analytes from tissue. Recently, laser postionization (MALDI-2) has been developed for increased ion yield and sensitivity for lipid IMS. However, the dependence of MALDI-2 performance on the various lipid classes is largely unknown. To understand the effect of the applied matrix on MALDI-2 analysis of lipids, samples including an equimolar lipid standard mixture, various tissue homogenates, and intact rat kidney tissue sections were analyzed using the following matrices: α-cyano-4-hydroxycinnamic acid, 2',5'-dihydroxyacetophenone, 2',5'-dihydroxybenzoic acid (DHB), and norharmane (NOR). Lipid signal enhancement of protonated species using MALDI-2 technology varied based on the matrix used. Although signal improvements were observed for all matrices, the most dramatic effects using MALDI-2 were observed using NOR and DHB. For lipid standards analyzed by MALDI-2, NOR provided the broadest coverage, enabling the detection of all 13 protonated standards, including nonpolar lipids, whereas DHB gave less coverage but gave the highest signal increase for those lipids recorded. With respect to tissue homogenates and rat kidney tissue, mass spectra were compared and showed that the number and intensity of neutral lipids tentatively identified with MALDI-2 using NOR increased significantly (e.g., fivefold intensity increase for triacylglycerol). In the cases of DHB with MALDI-2, the number of protonated lipids identified from tissue homogenates doubled with 152 on average compared with 76 with MALDI alone. High spatial resolution imaging (~20 μm) of rat kidney tissue showed similar results using DHB with 125 lipids tentatively identified from MALDI-2 spectra versus just 72 using standard MALDI. From the four matrices tested, NOR provided the greatest increase in sensitivity for neutral lipids (triacylglycerol, diacylglycerol, monoacylglycerol, and cholesterol ester), and DHB provided the highest overall number of lipids detected using MALDI-2 technology.
2020-12-01Integrating ion mobility and imaging mass spectrometry for comprehensive analysis of biological tissues: A brief review and perspectiveRivera ES, Djambazova KV, Neumann EK, Caprioli RM, Spraggins JMImaging mass spectrometry (IMS) technologies are capable of mapping a wide array of biomolecules in diverse cellular and tissue environments. IMS has emerged as an essential tool for providing spatially targeted molecular information due to its high sensitivity, wide molecular coverage, and chemical specificity. One of the major challenges for mapping the complex cellular milieu is the presence of many isomers and isobars in these samples. This challenge is traditionally addressed using orthogonal liquid chromatography (LC)-based analysis, though, common approaches such as chromatography and electrophoresis are not able to be performed at timescales that are compatible with most imaging applications. Ion mobility offers rapid, gas-phase separations that are readily integrated with IMS workflows in order to provide additional data dimensionality that can improve signal-to-noise, dynamic range, and specificity. Here, we highlight recent examples of ion mobility coupled to IMS and highlight their importance to the field.
2020-12-01Progenitor identification and SARS-CoV-2 infection in human distal lung organoidsSalahudeen AA, Choi SS, Rustagi A, Zhu J, van Unen V, de la O SM, Flynn RA, Margalef-Català M, Santos AJM, Ju J, Batish A, Usui T, Zheng GXY, Edwards CE, Wagar LE, Luca V, Anchang B, Nagendran M, Nguyen K, Hart DJ, Terry JM, Belgrader P, Ziraldo SB, Mikkelsen TS, Harbury PB, Glenn JS, Garcia KC, Davis MM, Baric RS, Sabatti C, Amieva MR, Blish CA, Desai TJ, Kuo CJ.TTD-StanfordThe distal lung contains terminal bronchioles and alveoli that facilitate gas exchange. Three-dimensional in vitro human distal lung culture systems would strongly facilitate the investigation of pathologies such as interstitial lung disease, cancer and coronavirus disease 2019 (COVID-19) pneumonia caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Here we describe the development of a long-term feeder-free, chemically defined culture system for distal lung progenitors as organoids derived from single adult human alveolar epithelial type II (AT2) or KRT5+ basal cells. AT2 organoids were able to differentiate into AT1 cells, and basal cell organoids developed lumens lined with differentiated club and ciliated cells. Single-cell analysis of KRT5+ cells in basal organoids revealed a distinct population of ITGA6+ITGB4+ mitotic cells, whose offspring further segregated into a TNFRSF12Ahi subfraction that comprised about ten per cent of KRT5+ basal cells. This subpopulation formed clusters within terminal bronchioles and exhibited enriched clonogenic organoid growth activity. We created distal lung organoids with apical-out polarity to present ACE2 on the exposed external surface, facilitating infection of AT2 and basal cultures with SARS-CoV-2 and identifying club cells as a target population. This long-term, feeder-free culture of human distal lung organoids, coupled with single-cell analysis, identifies functional heterogeneity among basal cells and establishes a facile in vitro organoid model of human distal lung infections, including COVID-19-associated pneumonia.
2020-12-02Lipid Landscape of the Human Retina and Supporting Tissues Revealed by High-Resolution Imaging Mass SpectrometryAnderson DMG, Messinger JD, Patterson NH, Rivera ES, Kotnala A, Spraggins JM, Caprioli RM, Curcio CA, Schey KL.TMC-Vanderbilt (Eye/pancreas)The human retina provides vision at light levels ranging from starlight to sunlight. Its supporting tissues regulate plasma-delivered lipophilic essentials for vision, including retinoids. The macula is an anatomic specialization for high-acuity and color vision that is also vulnerable to prevalent blinding diseases. The retina's exquisite architecture comprises numerous cell types that are aligned horizontally, yielding structurally distinct cell, synaptic, and vascular layers that are visible in histology and in diagnostic clinical imaging. MALDI imaging mass spectrometry (IMS) is now capable of uniting low micrometer spatial resolution with high levels of chemical specificity. In this study, a multimodal imaging approach fortified with accurate multi-image registration was used to localize lipids in human retina tissue at laminar, cellular, and subcellular levels. Multimodal imaging results indicate differences in distributions and abundances of lipid species across and within single cell types. Of note are distinct localizations of signals within specific layers of the macula. For example, phosphatidylethanolamine and phosphatidylinositol lipids were localized to central RPE cells, whereas specific plasmalogen lipids were localized to cells of the perifoveal RPE and Henle fiber layer. Subcellular compartments of photoreceptors were distinguished by PE(20:0_22:5) in the outer nuclear layer, PE(18:0_22:6) in outer and inner segments, and cardiolipin CL(70:5) in the mitochondria-rich inner segments. Several lipids, differing by a single double bond, have markedly different distributions between the central fovea and the ganglion cell and inner nuclear layers. A lipid atlas, initiated in this study, can serve as a reference database for future examination of diseased tissues.
2020-12-10GCNG: graph convolutional networks for inferring gene interaction from spatial transcriptomics dataYuan Y, Bar-Joseph ZHIVE TC-CMUMost methods for inferring gene-gene interactions from expression data focus on intracellular interactions. The availability of high-throughput spatial expression data opens the door to methods that can infer such interactions both within and between cells. To achieve this, we developed Graph Convolutional Neural networks for Genes (GCNG). GCNG encodes the spatial information as a graph and combines it with expression data using supervised training. GCNG improves upon prior methods used to analyze spatial transcriptomics data and can propose novel pairs of extracellular interacting genes. The output of GCNG can also be used for downstream analysis including functional gene assignment.Supporting website with software and data: .
2020-12-10High-Spatial-Resolution Multi-Omics Sequencing via Deterministic Barcoding in TissueLiu Y, Yang M, Deng Y, Su G, Enninful A, Guo CC, Tebaldi T, Zhang D, Kim D, Bai Z, Norris E, Pan A, Li J, Xiao Y, Halene S, Fan RTTD-YaleWe present deterministic barcoding in tissue for spatial omics sequencing (DBiT-seq) for co-mapping of mRNAs and proteins in a formaldehyde-fixed tissue slide via next-generation sequencing (NGS). Parallel microfluidic channels were used to deliver DNA barcodes to the surface of a tissue slide, and crossflow of two sets of barcodes, A1-50 and B1-50, followed by ligation in situ, yielded a 2D mosaic of tissue pixels, each containing a unique full barcode AB. Application to mouse embryos revealed major tissue types in early organogenesis as well as fine features like microvasculature in a brain and pigmented epithelium in an eye field. Gene expression profiles in 10-μm pixels conformed into the clusters of single-cell transcriptomes, allowing for rapid identification of cell types and spatial distributions. DBiT-seq can be adopted by researchers with no experience in microfluidics and may find applications in a range of fields including developmental biology, cancer biology, neuroscience, and clinical pathology.
2021-01-27Integrated spatial genomics reveals global architecture of single nucleiTakei Y, Yun J, Zheng S, Ollikainen N, Pierson N, White J, Shah S, Thomassie J, Suo S, Eng CL, Guttman M, Yuan GC, Cai L.TMC-Cal TechIdentifying the relationships between chromosome structures, nuclear bodies, chromatin states and gene expression is an overarching goal of nuclear-organization studies1-4. Because individual cells appear to be highly variable at all these levels5, it is essential to map different modalities in the same cells. Here we report the imaging of 3,660 chromosomal loci in single mouse embryonic stem (ES) cells using DNA seqFISH+, along with 17 chromatin marks and subnuclear structures by sequential immunofluorescence and the expression profile of 70 RNAs. Many loci were invariably associated with immunofluorescence marks in single mouse ES cells. These loci form 'fixed points' in the nuclear organizations of single cells and often appear on the surfaces of nuclear bodies and zones defined by combinatorial chromatin marks. Furthermore, highly expressed genes appear to be pre-positioned to active nuclear zones, independent of bursting dynamics in single cells. Our analysis also uncovered several distinct mouse ES cell subpopulations with characteristic combinatorial chromatin states. Using clonal analysis, we show that the global levels of some chromatin marks, such as H3 trimethylation at lysine 27 (H3K27me3) and macroH2A1 (mH2A1), are heritable over at least 3-4 generations, whereas other marks fluctuate on a faster time scale. This seqFISH+-based spatial multimodal approach can be used to explore nuclear organization and cell states in diverse biological systems.
2021-01-31Predictive modeling of single-cell DNA methylome data enhances integration with transcriptome dataUzun Y, Wu H, Tan K.TMC-CHOPSingle-cell DNA methylation data has become increasingly abundant and has uncovered many genes with a positive correlation between expression and promoter methylation, challenging the common dogma based on bulk data. However, computational tools for analyzing single-cell methylome data are lagging far behind. A number of tasks, including cell type calling and integration with transcriptome data, requires the construction of a robust gene activity matrix as the prerequisite but challenging task. The advent of multi-omics data enables measurement of both DNA methylation and gene expression for the same single cells. Although such data is rather sparse, they are sufficient to train supervised models that capture the complex relationship between DNA methylation and gene expression and predict gene activities at single-cell level. Here, we present methylome association by predictive linkage to expression (MAPLE), a computational framework that learns the association between DNA methylation and expression using both gene- and cell-dependent statistical features. Using multiple data sets generated with different experimental protocols, we show that using predicted gene activity values significantly improves several analysis tasks, including clustering, cell type identification, and integration with transcriptome data. Application of MAPLE revealed several interesting biological insights into the relationship between methylation and gene expression, including asymmetric importance of methylation signals around transcription start site for predicting gene expression, and increased predictive power of methylation signals in promoters located outside CpG islands and shores. With the rapid accumulation of single-cell epigenomics data, MAPLE provides a general framework for integrating such data with transcriptome data.
2021-02-15Construction of a Multi-Phase Contrast Computed Tomography Kidney AtlasLee HH, Tang Y, Xu K, Bao S, Fogo AB, Harris R, de Caestecker MP, Heinrich M, Spraggins JM, Huo Y, Landman BATMC-Vanderbilt (Kidney)The Human BioMolecular Atlas Program (HuBMAP) seeks to create a molecular atlas at the cellular level of the human body to spur interdisciplinary innovations across spatial and temporal scales. While the preponderance of effort is allocated towards cellular and molecular scale mapping, differentiating and contextualizing findings within tissues, organs and systems are essential for the HuBMAP efforts. The kidney is an initial organ target of HuBMAP, and constructing a framework (or atlas) for integrating information across scales is needed for visualizing and integrating information. However, there is no abdominal atlas currently available in the public domain. Substantial variation in healthy kidneys exists with sex, body size, and imaging protocols. With the integration of clinical archives for secondary research use, we are able to build atlases based on a diverse population and clinically relevant protocols. In this study, we created a computed tomography (CT) phase-specific atlas for the abdomen, which is optimized for the kidney organ. A two-stage registration pipeline was used by registering extracted abdominal volume of interest from body part regression, to a high-resolution CT. Affine and non-rigid registration were performed to all scans hierarchically. To generate and evaluate the atlas, multiphase CT scans of 500 control subjects (age: 15 - 50, 250 males, 250 females) are registered to the atlas target through the complete pipeline. The abdominal body and kidney registration are shown to be stable with the variance map computed from the result average template. Both left and right kidneys are substantially localized in the high-resolution target space, which successfully demonstrated the sharp details of its anatomical characteristics across each phase. We illustrated the applicability of the atlas template for integrating across normal kidney variation from 64 cm3 to 302 cm3.
2021-02-15Renal Cortex, Medulla and Pelvicaliceal System Segmentation on Arterial Phase CT Images with Random Patch-based NetworksTang Y, Gao R, Lee HH, Xu Z, Savoie BV, Bao S, Huo Y, Fogo AB, Harris R, de Caestecker MP, Spraggins J, Landman BATMC-Vanderbilt (Kidney)Renal segmentation on contrast-enhanced computed tomography (CT) provides distinct spatial context and morphology. Current studies for renal segmentations are highly dependent on manual efforts, which are time-consuming and tedious. Hence, developing an automatic framework for the segmentation of renal cortex, medulla and pelvicalyceal system is an important quantitative assessment of renal morphometry. Recent innovations in deep methods have driven performance toward levels for which clinical translation is appealing. However, the segmentation of renal structures can be challenging due to the limited field-of-view (FOV) and variability among patients. In this paper, we propose a method to automatically label the renal cortex, the medulla and pelvicalyceal system. First, we retrieved 45 clinically-acquired deidentified arterial phase CT scans (45 patients, 90 kidneys) without diagnosis codes (ICD-9) involving kidney abnormalities. Second, an interpreter performed manual segmentation to pelvis, medulla and cortex slice-by-slice on all retrieved subjects under expert supervision. Finally, we proposed a patch-based deep neural networks to automatically segment renal structures. Compared to the automatic baseline algorithm (3D U-Net) and conventional hierarchical method (3D U-Net Hierarchy), our proposed method achieves improvement of 0.7968 to 0.6749 (3D U-Net), 0.7482 (3D U-Net Hierarchy) in terms of mean Dice scores across three classes (p-value < 0.001, paired t-tests between our method and 3D U-Net Hierarchy). In summary, the proposed algorithm provides a precise and efficient method for labeling renal structures.
2021-02-23Spatial Segmentation of Mass Spectrometry Imaging Data by Combining Multivariate Clustering and Univariate Thresholding.Hu H, Yin R, Brown HM, Laskin JTTD-PurdueSpatial segmentation partitions mass spectrometry imaging (MSI) data into distinct regions, providing a concise visualization of the vast amount of data and identifying regions of interest (ROIs) for downstream statistical analysis. Unsupervised approaches are particularly attractive, as they may be used to discover the underlying subpopulations present in the high-dimensional MSI data without prior knowledge of the properties of the sample. Herein, we introduce an unsupervised spatial segmentation approach, which combines multivariate clustering and univariate thresholding to generate comprehensive spatial segmentation maps of the MSI data. This approach combines matrix factorization and manifold learning to enable high-quality image segmentation without an extensive hyperparameter search. In parallel, some ion images inadequately represented in the multivariate analysis were treated using univariate thresholding to generate complementary spatial segments. The final spatial segmentation map was assembled from segment candidates that were generated using both techniques. We demonstrate the performance and robustness of this approach for two MSI data sets of mouse uterine and kidney tissue sections that were acquired with different spatial resolutions. The resulting segmentation maps are easy to interpret and project onto the known anatomical regions of the tissue.
2021-03-01Surfactant-assisted one-pot sample preparation for label-free single-cell proteomicsTsai, CF., Zhang, P., Scholten, D. et al.TTD-PNNLLarge numbers of cells are generally required for quantitative global proteome profiling due to surface adsorption losses associated with sample processing. Such bulk measurement obscures important cell-to-cell variability (cell heterogeneity) and makes proteomic profiling impossible for rare cell populations (e.g., circulating tumor cells (CTCs)). Here we report a surfactant-assisted one-pot sample preparation coupled with mass spectrometry (MS) method termed SOP-MS for label-free global single-cell proteomics. SOP-MS capitalizes on the combination of a MS-compatible nonionic surfactant, n-Dodecyl-β-D-maltoside, and hydrophobic surface-based low-bind tubes or multi-well plates for ‘all-in-one’ one-pot sample preparation. This ‘all-in-one’ method including elimination of all sample transfer steps maximally reduces surface adsorption losses for effective processing of single cells, thus improving detection sensitivity for single-cell proteomics. This method allows convenient label-free quantification of hundreds of proteins from single human cells and ~1200 proteins from small tissue sections (close to ~20 cells). When applied to a patient CTC-derived xenograft (PCDX) model at the single-cell resolution, SOP-MS can reveal distinct protein signatures between primary tumor cells and early metastatic lung cells, which are related to the selection pressure of anti-tumor immunity during breast cancer metastasis. The approach paves the way for routine, precise, quantitative single-cell proteomics.
2021-03-08Giotto: a toolbox for integrative analysis and visualization of spatial expression dataDries R, Zhu Q, Dong R, Eng CL, Li H, Liu K, Fu Y, Zhao T, Sarkar A, Bao F, George RE, Pierson N, Cai L, Yuan GC.TTD-Cal TechSpatial transcriptomic and proteomic technologies have provided new opportunities to investigate cells in their native microenvironment. Here we present Giotto, a comprehensive and open-source toolbox for spatial data analysis and visualization. The analysis module provides end-to-end analysis by implementing a wide range of algorithms for characterizing tissue composition, spatial expression patterns, and cellular interactions. Furthermore, single-cell RNAseq data can be integrated for spatial cell-type enrichment analysis. The visualization module allows users to interactively visualize analysis outputs and imaging features. To demonstrate its general applicability, we apply Giotto to a wide range of datasets encompassing diverse technologies and platforms.
2021-04-20Quantitative Mass Spectrometry Imaging of Biological SystemsUnsihuay D, Mesa Sanchez D, Laskin J.TTD-PurdueMass spectrometry imaging (MSI) is a powerful, label-free technique that provides detailed maps of hundreds of molecules in complex samples with high sensitivity and subcellular spatial resolution. Accurate quantification in MSI relies on a detailed understanding of matrix effects associated with the ionization process along with evaluation of the extraction efficiency and mass-dependent ion losses occurring in the analysis step. We present a critical summary of approaches developed for quantitative MSI of metabolites, lipids, and proteins in biological tissues and discuss their current and future applications.
2021-04-27Deeper Protein Identification Using Field Asymmetric Ion Mobility Spectrometry in Top-Down ProteomicsGerbasi VR, Melani RD, Abbatiello SE, Belford MW, Huguet R, McGee JP, Dayhoff D, Thomas PM, Kelleher NLRTI-NorthwesternField asymmetric ion mobility spectrometry (FAIMS), when used in proteomics studies, provides superior selectivity and enables more proteins to be identified by providing additional gas-phase separation. Here, we tested the performance of cylindrical FAIMS for the identification and characterization of proteoforms by top-down mass spectrometry of heterogeneous protein mixtures. Combining FAIMS with chromatographic separation resulted in a 62% increase in protein identifications, an 8% increase in proteoform identifications, and an improvement in proteoform identification compared to samples analyzed without FAIMS. In addition, utilization of FAIMS resulted in the identification of proteins encoded by lower-abundance mRNA transcripts. These improvements were attributable, in part, to improved signal-to-noise for proteoforms with similar retention times. Additionally, our results show that the optimal compensation voltage of any given proteoform was correlated with the molecular weight of the analyte. Collectively these results suggest that the addition of FAIMS can enhance top-down proteomics in both discovery and targeted applications.
2021-05-01Highly multiplexed tissue imaging using repeated oligonucleotide exchange reactionKennedy-Darling J, Bhate SS, Hickey JW, Black S, Barlow GL, Vazquez G, Venkataraaman VG, Samusik N, Goltsev Y, Schürch CM, Nolan GP.TMC-StanfordMultiparameter tissue imaging enables analysis of cell-cell interactions in situ, the cellular basis for tissue structure, and novel cell types that are spatially restricted, giving clues to biological mechanisms behind tissue homeostasis and disease. Here, we streamlined and simplified the multiplexed imaging method CO-Detection by indEXing (CODEX) by validating 58 unique oligonucleotide barcodes that can be conjugated to antibodies. We showed that barcoded antibodies retained their specificity for staining cognate targets in human tissue. Antibodies were visualized one at a time by adding a fluorescently labeled oligonucleotide complementary to oligonucleotide barcode, imaging, stripping, and repeating this cycle. With this we developed a panel of 46 antibodies that was used to stain five human lymphoid tissues: three tonsils, a spleen, and a LN. To analyze the data produced, an image processing and analysis pipeline was developed that enabled single-cell analysis on the data, including unsupervised clustering, that revealed 31 cell types across all tissues. We compared cell-type compositions within and directly surrounding follicles from the different lymphoid organs and evaluated cell-cell density correlations. This sequential oligonucleotide exchange technique enables a facile imaging of tissues that leverages pre-existing imaging infrastructure to decrease the barriers to broad use of multiplexed imaging.
2021-05-10SpatialDWLS: accurate deconvolution of spatial transcriptomic dataDong R, Yuan GCTTD-Cal TechRecent development of spatial transcriptomic technologies has made it possible to characterize cellular heterogeneity with spatial information. However, the technology often does not have sufficient resolution to distinguish neighboring cell types. Here, we present spatialDWLS, to quantitatively estimate the cell-type composition at each spatial location. We benchmark the performance of spatialDWLS by comparing it with a number of existing deconvolution methods and find that spatialDWLS outperforms the other methods in terms of accuracy and speed. By applying spatialDWLS to a human developmental heart dataset, we observe striking spatial temporal changes of cell-type composition during development.
2021-05-17Identifying signaling genes in spatial single-cell expression dataLi D, Ding J, Bar-Joseph ZHIVE TC-CMUMotivation: Recent technological advances enable the profiling of spatial single-cell expression data. Such data present a unique opportunity to study cell-cell interactions and the signaling genes that mediate them. However, most current methods for the analysis of these data focus on unsupervised descriptive modeling, making it hard to identify key signaling genes and quantitatively assess their impact. Results: We developed a Mixture of Experts for Spatial Signaling genes Identification (MESSI) method to identify active signaling genes within and between cells. The mixture of experts strategy enables MESSI to subdivide cells into subtypes. MESSI relies on multi-task learning using information from neighboring cells to improve the prediction of response genes within a cell. Applying the methods to three spatial single-cell expression datasets, we show that MESSI accurately predicts the levels of response genes, improving upon prior methods and provides useful biological insights about key signaling genes and subtypes of excitatory neuron cells. Availability and implementation: MESSI is available at: Supplementary information: Supplementary data are available at Bioinformatics online.
2021-06-07The emerging landscape of single-molecule protein sequencing technologies.Alfaro JA, Bohländer P, Dai M, Filius M, Howard CJ, van Kooten XF, Ohayon S, Pomorski A, Schmid S, Aksimentiev A, Anslyn EV, Bedran G, Cao C, Chinappi M, Coyaud E, Dekker C, Dittmar G, Drachman N, Eelkema R, Goodlett D, Hentz S, Kalathiya U, Kelleher NL, Kelly RT, Kelman Z, Kim SH, Kuster B, Rodriguez-Larrea D, Lindsay S, Maglia G, Marcotte EM, Marino JP, Masselon C, Mayer M, Samaras P, Sarthak K, Sepiashvili L, Stein D, Wanunu M, Wilhelm M, Yin P, Meller A, Joo CRTI-NorthwesternSingle-cell profiling methods have had a profound impact on the understanding of cellular heterogeneity. While genomes and transcriptomes can be explored at the single-cell level, single-cell profiling of proteomes is not yet established. Here we describe new single-molecule protein sequencing and identification technologies alongside innovations in mass spectrometry that will eventually enable broad sequence coverage in single-cell profiling. These technologies will in turn facilitate biological discovery and open new avenues for ultrasensitive disease diagnostics.
2021-06-08Multiomics Imaging Using High-Energy Water Gas Cluster Ion Beam Secondary Ion Mass Spectrometry [(H 2 O) n-GCIB-SIMS] of Frozen-Hydrated Cells and TissueTian H, Sheraz Née Rabbani S, Vickerman JC, Winograd NTTD-Columbia/Penn StateIntegration of multiomics at the single-cell level allows the unambiguous dissecting of phenotypic heterogeneity at different states such as health, disease, and biomedical response. Imaging mass spectrometry holds the promise of being able to measure multiple types of biomolecules in parallel in the same cell. We have explored the possibility of using water gas cluster ion beam secondary ion mass spectrometry [(H2O)n-GCIB-SIMS] as an analytical tool for multiomics assay. (H2O)n-GCIB has been hailed as an ideal ionization source for biological sampling owing to the enhanced chemical sensitivity and reduced matrix effect. Taking advantage of 1 μm spatial resolution by using a high-energy beam system, we have clearly shown the enhancement of multiple intact biomolecules up to a few hundredfold in single cells. Coupled with the cryogenic sample preparation/measurement, the lipids and metabolites were imaged simultaneously within the cellular region, uncovering the pristine chemistry for integrated omics in the same sample. We have demonstrated that double-charged myelin protein fragments and single-charged multiple lipids and metabolites can be localized in the same cells/tissue with a single acquisition. Our exploration has also been extended to the capability of (H2O)n-GCIB in the generation of multiple charged peptides on protein standards. Frozen hydration combined with (H2O)n-GCIB provides the possibility of universal enhancement for the ionization of multiple bio-molecules, including peptides/proteins which has allowed "omics" to become feasible in the same sample using SIMS.
2021-06-24Integrated analysis of multimodal single-cell dataHao Y, Hao S, Andersen-Nissen E, Mauck WM 3rd, Zheng S, Butler A, Lee MJ, Wilk AJ, Darby C, Zager M, Hoffman P, Stoeckius M, Papalexi E, Mimitou EP, Jain J, Srivastava A, Stuart T, Fleming LM, Yeung B, Rogers AJ, McElrath JM, Blish CA, Gottardo R, Smibert P, Satija RHIVE MC-NYGCThe simultaneous measurement of multiple modalities represents an exciting frontier for single-cell genomics and necessitates computational methods that can define cellular states based on multimodal data. 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 211,000 human peripheral blood mononuclear cells (PBMCs) with panels extending to 228 antibodies to construct a multimodal reference atlas of the circulating immune system. Multimodal analysis substantially improves our ability to resolve cell states, allowing us to identify and validate 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 coronavirus disease 2019 (COVID-19). Our approach represents a broadly applicable strategy to analyze single-cell multimodal datasets and to look beyond the transcriptome toward a unified and multimodal definition of cellular identity.
2021-07-02Embryo-scale, single-cell spatial transcriptomicsSrivatsan SR, Regier MC, Barkan E, Franks JM, Packer JS, Grosjean P, Duran M, Saxton S, Ladd JJ, Spielmann M, Lois C, Lampe PD, Shendure J, Stevens KR, Trapnell CTMC-Cal TechSpatial patterns of gene expression manifest at scales ranging from local (e.g., cell-cell interactions) to global (e.g., body axis patterning). However, current spatial transcriptomics methods either average local contexts or are restricted to limited fields of view. Here, we introduce sci-Space, which retains single-cell resolution while resolving spatial heterogeneity at larger scales. Applying sci-Space to developing mouse embryos, we captured approximate spatial coordinates and whole transcriptomes of about 120,000 nuclei. We identify thousands of genes exhibiting anatomically patterned expression, leverage spatial information to annotate cellular subtypes, show that cell types vary substantially in their extent of spatial patterning, and reveal correlations between pseudotime and the migratory patterns of differentiating neurons. Looking forward, we anticipate that sci-Space will facilitate the construction of spatially resolved single-cell atlases of mammalian development.
2021-07-08Mass spectrometry-based metabolomics: a guide for annotation, quantification and best reporting practicesAlseekh S, Aharoni A, Brotman Y, Contrepois K, D'Auria J, Ewald J, C Ewald J, Fraser PD, Giavalisco P, Hall RD, Heinemann M, Link H, Luo J, Neumann S, Nielsen J, Perez de Souza L, Saito K, Sauer U, Schroeder FC, Schuster S, Siuzdak G, Skirycz A, Sumner LW, Snyder MP, Tang H, Tohge T, Wang Y, Wen W, Wu S, Xu G, Zamboni N, Fernie ARTMC-StanfordMass spectrometry-based metabolomics approaches can enable detection and quantification of many thousands of metabolite features simultaneously. However, compound identification and reliable quantification are greatly complicated owing to the chemical complexity and dynamic range of the metabolome. Simultaneous quantification of many metabolites within complex mixtures can additionally be complicated by ion suppression, fragmentation and the presence of isomers. Here we present guidelines covering sample preparation, replication and randomization, quantification, recovery and recombination, ion suppression and peak misidentification, as a means to enable high-quality reporting of liquid chromatography- and gas chromatography-mass spectrometry-based metabolomics-derived data.
2021-08-02CODEX multiplexed tissue imaging with DNA-conjugated antibodiesBlack S, Phillips D, Hickey JW, Kennedy-Darling J, Venkataraaman VG, Samusik N, Goltsev Y, Schürch CM, Nolan GPTMC-StanfordAdvances in multiplexed imaging technologies have drastically improved our ability to characterize healthy and diseased tissues at the single-cell level. Co-detection by indexing (CODEX) relies on DNA-conjugated antibodies and the cyclic addition and removal of complementary fluorescently labeled DNA probes and has been used so far to simultaneously visualize up to 60 markers in situ. CODEX enables a deep view into the single-cell spatial relationships in tissues and is intended to spur discovery in developmental biology, disease and therapeutic design. Herein, we provide optimized protocols for conjugating purified antibodies to DNA oligonucleotides, validating the conjugation by CODEX staining and executing the CODEX multicycle imaging procedure for both formalin-fixed, paraffin-embedded (FFPE) and fresh-frozen tissues. In addition, we describe basic image processing and data analysis procedures. We apply this approach to an FFPE human tonsil multicycle experiment. The hands-on experimental time for antibody conjugation is ~4.5 h, validation of DNA-conjugated antibodies with CODEX staining takes ~6.5 h and preparation for a CODEX multicycle experiment takes ~8 h. The multicycle imaging and data analysis time depends on the tissue size, number of markers in the panel and computational complexity.
2021-08-10Immunophenotyping assessment in a COVID-19 cohort (IMPACC): A prospective longitudinal studyIMPACC Manuscript Writing Team; IMPACC Network Steering CommitteeTMC-FloridaThe IMmunoPhenotyping Assessment in a COVID-19 Cohort (IMPACC) is a prospective longitudinal study designed to enroll 1000 hospitalized patients with COVID-19 (NCT04378777). IMPACC collects detailed clinical, laboratory and radiographic data along with longitudinal biologic sampling of blood and respiratory secretions for in depth testing. Clinical and lab data are integrated to identify immunologic, virologic, proteomic, metabolomic and genomic features of COVID-19-related susceptibility, severity and disease progression. The goals of IMPACC are to better understand the contributions of pathogen dynamics and host immune responses to the severity and course of COVID-19 and to generate hypotheses for identification of biomarkers and effective therapeutics, including optimal timing of such interventions. In this report we summarize the IMPACC study design and protocols including clinical criteria and recruitment, multi-site standardized sample collection and processing, virologic and immunologic assays, harmonization of assay protocols, high-level analyses and the data sharing plans.
2021-08-27α-Cyano-4-hydroxycinnamic Acid and Tri-Potassium Citrate Salt Pre-Coated Silicon Nanopost Array Provides Enhanced Lipid Detection for High Spatial Resolution MALDI Imaging Mass SpectrometryDufresne M, Fincher JA, Patterson NH, Schey KL, Norris JL, Caprioli RM, Spraggins JMTMC-Vanderbilt (Eye/pancreas)We have developed a pre-coated substrate for matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry (IMS) that enables high spatial resolution mapping of both phospholipids and neutral lipid classes in positive ion mode as metal cation adducts. The MALDI substrates are constructed by depositing a layer of α-cyano-4-hydroxycinnamic acid (CHCA) and potassium salts onto silicon nanopost arrays (NAPA) prior to tissue mounting. The matrix/salt pre-coated NAPA substrate significantly enhances all detected lipid signals allowing lipids to be detected at lower laser energies than bare NAPA. The improved sensitivity at lower laser energy enabled ion images to be generated at 10 μm spatial resolution from rat retinal tissue. Optimization of matrix pre-coated NAPA consisted of testing lithium, sodium, and potassium salts along with various matrices to investigate the increased sensitivity toward lipids for MALDI IMS experiments. It was determined that pre-coating NAPA with CHCA and potassium salts before thaw-mounting of tissue resulted in a signal intensity increase of at least 5.8 ± 0.1-fold for phospholipids and 2.0 ± 0.1-fold for neutral lipids compared to bare NAPA. Pre-coating NAPA with matrix and salt also reduced the necessary laser power to achieve desorption/ionization by ∼35%. This reduced the effective diameter of the ablation area from 13 ± 2 μm down to 8 ± 1 μm, enabling high spatial resolution MALDI IMS. Using pre-coated NAPA with CHCA and potassium salts offers a MALDI IMS substrate with broad molecular coverage of lipids in a single polarity that eliminates the need for extensive sample preparation after sectioning.
2021-09-06Integration of spatial and single-cell transcriptomic data elucidates mouse organogenesisLohoff T, Ghazanfar S, Missarova A, Koulena N, Pierson N, Griffiths JA, Bardot ES, Eng CL, Tyser RCV, Argelaguet R, Guibentif C, Srinivas S, Briscoe J, Simons BD, Hadjantonakis AK, Göttgens B, Reik W, Nichols J, Cai L, Marioni JCHIVE MC-NYGCMolecular profiling of single cells has advanced our knowledge of the molecular basis of development. However, current approaches mostly rely on dissociating cells from tissues, thereby losing the crucial spatial context of regulatory processes. Here, we apply an image-based single-cell transcriptomics method, sequential fluorescence in situ hybridization (seqFISH), to detect mRNAs for 387 target genes in tissue sections of mouse embryos at the 8-12 somite stage. By integrating spatial context and multiplexed transcriptional measurements with two single-cell transcriptome atlases, we characterize cell types across the embryo and demonstrate that spatially resolved expression of genes not profiled by seqFISH can be imputed. We use this high-resolution spatial map to characterize fundamental steps in the patterning of the midbrain-hindbrain boundary (MHB) and the developing gut tube. We uncover axes of cell differentiation that are not apparent from single-cell RNA-sequencing (scRNA-seq) data, such as early dorsal-ventral separation of esophageal and tracheal progenitor populations in the gut tube. Our method provides an approach for studying cell fate decisions in complex tissues and development.
2021-10-14Scalable dual-omics profiling with single-nucleus chromatin accessibility and mRNA expression sequencing 2 (SNARE-seq2)Plongthongkum N, Diep D, Chen S, Lake BB, Zhang K.TMC-UCSDComprehensive characterization of cellular heterogeneity and the underlying regulatory landscapes of tissues and organs requires a highly robust and scalable method to acquire matched RNA and chromatin accessibility profiles on the same cells. Here, we describe a single-nucleus chromatin accessibility and mRNA expression sequencing 2 (SNARE-seq2) assay, implemented with cellular combinatorial indexing. This method involves tagmentation within permeabilized and fixed single-nucleus isolates to capture accessible chromatin (AC) regions, followed by the capture and reverse transcription of RNA transcripts. Through combinatorial split pool ligations, cDNA and AC within each single nucleus become appended with a common cell barcode combination. The captured cDNA and AC are then co-amplified before splitting and enrichment into single-nucleus RNA and single-nucleus AC sequencing libraries. This protocol is compatible with both nuclei and whole cells and can be completed in 3.5 d. SNARE-seq2 permits robust generation of high-quality, joint single-cell RNA and AC sequencing libraries from hundreds of thousands of single cells per experiment.
2021-11-01In-depth triacylglycerol profiling using MS3 Q-Trap mass spectrometry.Cabruja M, Priotti J, Domizi P, Papsdorf K, Kroetz DL, Brunet A, Contrepois K, Snyder MPTMC-StanfordTotal triacylglycerol (TAG) level is a key clinical marker of metabolic and cardiovascular diseases. However, the roles of individual TAGs have not been thoroughly explored in part due to their extreme structural complexity. We present a targeted mass spectrometry-based method combining multiple reaction monitoring (MRM) and multiple stage mass spectrometry (MS3) for the comprehensive qualitative and semiquantitative profiling of TAGs. This method referred as TriP-MS3 - triacylglycerol profiling using MS3 - screens for more than 6,700 TAG species in a fully automated fashion. TriP-MS3 demonstrated excellent reproducibility (median interday CV ∼ 0.15) and linearity (median R2 = 0.978) and detected 285 individual TAG species in human plasma. The semiquantitative accuracy of the method was validated by comparison with a state-of-the-art reverse phase liquid chromatography (RPLC)-MS (R2 = 0.83), which is the most commonly used approach for TAGs profiling. Finally, we demonstrate the utility and the versatility of the method by characterizing the effects of a fatty acid desaturase inhibitor on TAG profiles in vitro and by profiling TAGs in Caenorhabditis elegans.

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