scholarly journals Multiplexed PCR-Free Detection of MicroRNAs in Single Cancer Cells Using a DNA-Barcoded Microtrough Array Chip

Micromachines ◽  
2019 ◽  
Vol 10 (4) ◽  
pp. 215 ◽  
Author(s):  
Nayi Wang ◽  
Yao Lu ◽  
Zhuo Chen ◽  
Rong Fan
Keyword(s):  
Single Cell ◽  
High Throughput ◽  
Single Cells ◽  
Pcr Amplification ◽  
Intratumor Heterogeneity ◽  
Rna Molecules ◽  
Multiplexed Pcr ◽  
Microrna Detection ◽  
Wide Range ◽  
Health And Disease

MicroRNAs are a class of small RNA molecules that regulate the expression of mRNAs in a wide range of biological processes and are implicated in human health and disease such as cancers. How to measure microRNA profiles in single cells with high throughput is essential to the development of cell-based assays for interrogating microRNA-mediated intratumor heterogeneity and the design of new lab tests for diagnosis and monitoring of cancers. Here, we report on an in situ hybridization barcoding workflow implemented in a sub-nanoliter microtrough array chip for high-throughput and multiplexed microRNA detection at the single cell level. The microtroughs are used to encapsulate single cells that are fixed, permeabilized, and pre-incubated with microRNA detection probes, each of which consists of a capture strand complementary to specific microRNA and a unique reporter strand that can be photocleaved in the microtroughs and subsequently detected by an array of DNA barcodes patterned on the bottom of the microtroughs. In this way, the measurement of reporter strands released from single cells is a surrogate for detecting single-cell microRNA profiles. This approach permits direct measurement of microRNAs without PCR amplification owing to the small volume (<1 nL) of microtroughs. It offers high throughput and high multiplexing capability for evaluating microRNA heterogeneity in single cells, representing a new approach toward microRNA-based diagnosis and monitoring of complex human diseases.

scholarly journals Highly Multiplexed Single-Cell Full-Length cDNA Sequencing of human immune cells with 10X Genomics and R2C2

2020 ◽  
Author(s):  
Roger Volden ◽  
Christopher Vollmers
Keyword(s):  
B Cell ◽  
Single Cell ◽  
High Throughput ◽  
Cell Biology ◽  
Mononuclear Cells ◽  
Single Cells ◽  
Cell Receptor ◽  
Full Length ◽  
Full Length Cdna ◽  
Wide Range

AbstractSingle cell transcriptome analysis elucidates facets of cell biology that have been previously out of reach. However, the high-throughput analysis of thousands of single cell transcriptomes has been limited by sample preparation and sequencing technology. High-throughput single cell analysis today is facilitated by protocols like the 10X Genomics platform or Drop-Seq which generate cDNA pools in which the origin of a transcript is encoded at its 5’ or 3’ end. These cDNA pools are currently analyzed by short read Illumina sequencing which can identify the cellular origin of a transcript and what gene it was transcribed from. However, these methods fail to retrieve isoform information. In principle, cDNA pools prepared using these approaches can be analyzed with Pacific Biosciences and Oxford Nanopore long-read sequencers to retrieve isoform information but all current implementations rely heavily on Illumina short-reads for the analysis in addition to long reads. Here, we used R2C2 to sequence and demultiplex 9 million full-length cDNA molecules generated by the 10X Chromium platform from ∼3000 peripheral blood mononuclear cells (PBMCs). We used these reads to – independent from Illumina data – cluster cells into B cells, T cells, and Monocytes and generate isoform-level transcriptomes for these cell-types. We also generated isoform-level transcriptomes for all single cells and used this information to identify a wide range of isoform diversity between genes. Finally, we also designed a computational workflow to extract paired adaptive immune receptor – T cell receptor and B cell receptor (TCR and BCR) –sequences unique to each T and B cell. This work represents a new, simple, and powerful approach that –using a single sequencing method – can extract an unprecedented amount of information from thousands of single cells.

scholarly journals Single-Cell Multiomics Analysis for Drug Discovery

Metabolites ◽  
2021 ◽  
Vol 11 (11) ◽  
pp. 729
Author(s):  
Sam F. Nassar ◽  
Khadir Raddassi ◽  
Terence Wu
Keyword(s):  
Drug Discovery ◽  
Single Cell ◽  
Single Cell Analysis ◽  
Single Cells ◽  
Population Level ◽  
Single Species ◽  
Cell Populations ◽  
Omics Technologies ◽  
Rna Molecules ◽  
Health And Disease

Given the heterogeneity seen in cell populations within biological systems, analysis of single cells is necessary for studying mechanisms that cannot be identified on a bulk population level. There are significant variations in the biological and physiological function of cell populations due to the functional differences within, as well as between, single species as a result of the specific proteome, transcriptome, and metabolome that are unique to each individual cell. Single-cell analysis proves crucial in providing a comprehensive understanding of the biological and physiological properties underlying human health and disease. Omics technologies can help to examine proteins (proteomics), RNA molecules (transcriptomics), and the chemical processes involving metabolites (metabolomics) in cells, in addition to genomes. In this review, we discuss the value of multiomics in drug discovery and the importance of single-cell multiomics measurements. We will provide examples of the benefits of applying single-cell omics technologies in drug discovery and development. Moreover, we intend to show how multiomics offers the opportunity to understand the detailed events which produce or prevent disease, and ways in which the separate omics disciplines complement each other to build a broader, deeper knowledge base.

scholarly journals Cryopreservation of human cancers conserves tumour heterogeneity for single-cell multi-omics analysis

2021 ◽  
Vol 13 (1) ◽  
Author(s):  
Sunny Z. Wu ◽  
Daniel L. Roden ◽  
Ghamdan Al-Eryani ◽  
Nenad Bartonicek ◽  
Kate Harvey ◽  
...  
Keyword(s):  
Single Cell ◽  
Rna Sequencing ◽  
High Throughput ◽  
Single Cells ◽  
Cellular Heterogeneity ◽  
Tumour Heterogeneity ◽  
Fresh Tissue ◽  
Human Cancers ◽  
Cryopreserved Cell ◽  
Single Cell Rna Sequencing

Abstract Background High throughput single-cell RNA sequencing (scRNA-Seq) has emerged as a powerful tool for exploring cellular heterogeneity among complex human cancers. scRNA-Seq studies using fresh human surgical tissue are logistically difficult, preclude histopathological triage of samples, and limit the ability to perform batch processing. This hindrance can often introduce technical biases when integrating patient datasets and increase experimental costs. Although tissue preservation methods have been previously explored to address such issues, it is yet to be examined on complex human tissues, such as solid cancers and on high throughput scRNA-Seq platforms. Methods Using the Chromium 10X platform, we sequenced a total of ~ 120,000 cells from fresh and cryopreserved replicates across three primary breast cancers, two primary prostate cancers and a cutaneous melanoma. We performed detailed analyses between cells from each condition to assess the effects of cryopreservation on cellular heterogeneity, cell quality, clustering and the identification of gene ontologies. In addition, we performed single-cell immunophenotyping using CITE-Seq on a single breast cancer sample cryopreserved as solid tissue fragments. Results Tumour heterogeneity identified from fresh tissues was largely conserved in cryopreserved replicates. We show that sequencing of single cells prepared from cryopreserved tissue fragments or from cryopreserved cell suspensions is comparable to sequenced cells prepared from fresh tissue, with cryopreserved cell suspensions displaying higher correlations with fresh tissue in gene expression. We showed that cryopreservation had minimal impacts on the results of downstream analyses such as biological pathway enrichment. For some tumours, cryopreservation modestly increased cell stress signatures compared to freshly analysed tissue. Further, we demonstrate the advantage of cryopreserving whole-cells for detecting cell-surface proteins using CITE-Seq, which is impossible using other preservation methods such as single nuclei-sequencing. Conclusions We show that the viable cryopreservation of human cancers provides high-quality single-cells for multi-omics analysis. Our study guides new experimental designs for tissue biobanking for future clinical single-cell RNA sequencing studies.

scholarly journals The Development of an Effective Bacterial Single-Cell Lysis Method Suitable for Whole Genome Amplification in Microfluidic Platforms

Micromachines ◽  
2018 ◽  
Vol 9 (8) ◽  
pp. 367 ◽  
Author(s):  
Yuguang Liu ◽  
Dirk Schulze-Makuch ◽  
Jean-Pierre de Vera ◽  
Charles Cockell ◽  
Thomas Leya ◽  
...  
Keyword(s):  
Single Cell ◽  
Single Cells ◽  
Bacterial Species ◽  
Cell Manipulation ◽  
Microfluidic Platforms ◽  
Gram Positive ◽  
Gram Negative ◽  
Genome Amplification ◽  
Single Cell Sequencing ◽  
Wide Range

Single-cell sequencing is a powerful technology that provides the capability of analyzing a single cell within a population. This technology is mostly coupled with microfluidic systems for controlled cell manipulation and precise fluid handling to shed light on the genomes of a wide range of cells. So far, single-cell sequencing has been focused mostly on human cells due to the ease of lysing the cells for genome amplification. The major challenges that bacterial species pose to genome amplification from single cells include the rigid bacterial cell walls and the need for an effective lysis protocol compatible with microfluidic platforms. In this work, we present a lysis protocol that can be used to extract genomic DNA from both gram-positive and gram-negative species without interfering with the amplification chemistry. Corynebacterium glutamicum was chosen as a typical gram-positive model and Nostoc sp. as a gram-negative model due to major challenges reported in previous studies. Our protocol is based on thermal and chemical lysis. We consider 80% of single-cell replicates that lead to >5 ng DNA after amplification as successful attempts. The protocol was directly applied to Gloeocapsa sp. and the single cells of the eukaryotic Sphaerocystis sp. and achieved a 100% success rate.

scholarly journals Mito Hacker: a set of tools to enable high-throughput analysis of mitochondrial network morphology

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Ali Rohani ◽  
Jennifer A. Kashatus ◽  
Dane T. Sessions ◽  
Salma Sharmin ◽  
David F. Kashatus
Keyword(s):  
Single Cell ◽  
High Throughput ◽  
Data Science ◽  
Structural Information ◽  
Morphological Changes ◽  
Mitochondrial Morphology ◽  
Single Cell Level ◽  
Mitochondrial Network ◽  
Cell Level ◽  
Wide Range

Abstract Mitochondria are highly dynamic organelles that can exhibit a wide range of morphologies. Mitochondrial morphology can differ significantly across cell types, reflecting different physiological needs, but can also change rapidly in response to stress or the activation of signaling pathways. Understanding both the cause and consequences of these morphological changes is critical to fully understanding how mitochondrial function contributes to both normal and pathological physiology. However, while robust and quantitative analysis of mitochondrial morphology has become increasingly accessible, there is a need for new tools to generate and analyze large data sets of mitochondrial images in high throughput. The generation of such datasets is critical to fully benefit from rapidly evolving methods in data science, such as neural networks, that have shown tremendous value in extracting novel biological insights and generating new hypotheses. Here we describe a set of three computational tools, Cell Catcher, Mito Catcher and MiA, that we have developed to extract extensive mitochondrial network data on a single-cell level from multi-cell fluorescence images. Cell Catcher automatically separates and isolates individual cells from multi-cell images; Mito Catcher uses the statistical distribution of pixel intensities across the mitochondrial network to detect and remove background noise from the cell and segment the mitochondrial network; MiA uses the binarized mitochondrial network to perform more than 100 mitochondria-level and cell-level morphometric measurements. To validate the utility of this set of tools, we generated a database of morphological features for 630 individual cells that encode 0, 1 or 2 alleles of the mitochondrial fission GTPase Drp1 and demonstrate that these mitochondrial data could be used to predict Drp1 genotype with 87% accuracy. Together, this suite of tools enables the high-throughput and automated collection of detailed and quantitative mitochondrial structural information at a single-cell level. Furthermore, the data generated with these tools, when combined with advanced data science approaches, can be used to generate novel biological insights.

scholarly journals P02.10 FocuSCOPE: a single cell, multi-omics solution to simultaneously analyze tumor variants and microenvironment

2021 ◽  
Vol 9 (Suppl 1) ◽  
pp. A12.1-A12
Author(s):  
Y Arjmand Abbassi ◽  
N Fang ◽  
W Zhu ◽  
Y Zhou ◽  
Y Chen ◽  
...  
Keyword(s):  
Gene Expression ◽  
Tumor Microenvironment ◽  
Single Cell ◽  
High Throughput ◽  
Immune Cells ◽  
Genetic Variants ◽  
Expression Profiles ◽  
Single Cells ◽  
Gene Expression Profiles ◽  
Single Cell Sequencing

Recent advances of high-throughput single cell sequencing technologies have greatly improved our understanding of the complex biological systems. Heterogeneous samples such as tumor tissues commonly harbor cancer cell-specific genetic variants and gene expression profiles, both of which have been shown to be related to the mechanisms of disease development, progression, and responses to treatment. Furthermore, stromal and immune cells within tumor microenvironment interact with cancer cells to play important roles in tumor responses to systematic therapy such as immunotherapy or cell therapy. However, most current high-throughput single cell sequencing methods detect only gene expression levels or epigenetics events such as chromatin conformation. The information on important genetic variants including mutation or fusion is not captured. To better understand the mechanisms of tumor responses to systematic therapy, it is essential to decipher the connection between genotype and gene expression patterns of both tumor cells and cells in the tumor microenvironment. We developed FocuSCOPE, a high-throughput multi-omics sequencing solution that can detect both genetic variants and transcriptome from same single cells. FocuSCOPE has been used to successfully perform single cell analysis of both gene expression profiles and point mutations, fusion genes, or intracellular viral sequences from thousands of cells simultaneously, delivering comprehensive insights of tumor and immune cells in tumor microenvironment at single cell resolution.Disclosure InformationY. Arjmand Abbassi: None. N. Fang: None. W. Zhu: None. Y. Zhou: None. Y. Chen: None. U. Deutsch: None.

scholarly journals Multiplexed Profiling of Single-cell Extracellular Vesicles Secretion

2018 ◽  
Author(s):  
Yahui Ji ◽  
Dongyuan Qi ◽  
Linmei Li ◽  
Haoran Su ◽  
Xiaojie Li ◽  
...  
Keyword(s):  
Single Cell ◽  
Cell Lines ◽  
Extracellular Vesicles ◽  
Single Cells ◽  
Cell Communication ◽  
Deep Understanding ◽  
Surface Markers ◽  
Cell Heterogeneity ◽  
Health And Disease ◽  
Cytokines Secretion

AbstractExtracellular vesicles (EVs) are important intercellular mediators regulating health and disease. Conventional EVs surface marker profiling, which was based on population measurements, masked the cell-to-cell heterogeneity in the quantity and phenotypes of EVs secretion. Herein, by using spatially patterned antibodies barcode, we realized multiplexed profiling of single-cell EVs secretion from more than 1000 single cells simultaneously. Applying this platform to profile human oral squamous cell carcinoma (OSCC) cell lines led to deep understanding of previously undifferentiated single cell heterogeneity underlying EVs secretion. Notably, we observed the decrement of certain EV phenotypes (e.g. CD63+EVs) were associated with the invasive feature of both OSCC cell lines and primary OSCC cells. We also realized multiplexed detection of EVs secretion and cytokines secretion simultaneously from the same single cells to investigate multidimensional spectrum of intercellular communications, from which we resolved three functional subgroups with distinct secretion profiles by visualized clustering. In particular, we found EVs secretion and cytokines secretion were generally dominated by different cell subgroups. The technology introduced here enables comprehensive evaluation of EVs secretion heterogeneity at single cell level, which may become an indispensable tool to complement current single cell analysis and EV research.SignificanceExtracellular vesicles (EVs) are cell derived nano-sized particles medicating cell-cell communication and transferring biology information molecules like nucleic acids to regulate human health and disease. Conventional methods for EV surface markers profiling can’t tell the differences in the quantity and phenotypes of EVs secretion between cells. To address this need, we developed a platform for profiling an array of surface markers on EVs from large numbers of single cells, enabling more comprehensive monitoring of cellular communications. Single cell EVs secretion assay led to previously unobserved cell heterogeneity underlying EVs secretion, which might open up new avenues for studying cell communication and cell microenvironment in both basic and clinical research.

scholarly journals Microfluidic guillotine for single-cell wound repair studies

2017 ◽  
Vol 114 (28) ◽  
pp. 7283-7288 ◽  
Author(s):  
Lucas R. Blauch ◽  
Ya Gai ◽  
Jian Wei Khor ◽  
Pranidhi Sood ◽  
Wallace F. Marshall ◽  
...  
Keyword(s):  
Single Cell ◽  
High Throughput ◽  
Wound Repair ◽  
Time Course ◽  
Single Cells ◽  
Repair Process ◽  
Gene Knockdown ◽  
Viscous Stress ◽  
Repair Capacity ◽  
Stentor Coeruleus

Wound repair is a key feature distinguishing living from nonliving matter. Single cells are increasingly recognized to be capable of healing wounds. The lack of reproducible, high-throughput wounding methods has hindered single-cell wound repair studies. This work describes a microfluidic guillotine for bisecting single Stentor coeruleus cells in a continuous-flow manner. Stentor is used as a model due to its robust repair capacity and the ability to perform gene knockdown in a high-throughput manner. Local cutting dynamics reveals two regimes under which cells are bisected, one at low viscous stress where cells are cut with small membrane ruptures and high viability and one at high viscous stress where cells are cut with extended membrane ruptures and decreased viability. A cutting throughput up to 64 cells per minute—more than 200 times faster than current methods—is achieved. The method allows the generation of more than 100 cells in a synchronized stage of their repair process. This capacity, combined with high-throughput gene knockdown in Stentor, enables time-course mechanistic studies impossible with current wounding methods.

The JAK2V617F Stem Cell Burden Is Mosaic, Dynamic and Correlates with Clinical Disease Burden in the MPN: An Exploitable Target

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 4090-4090
Author(s):  
Alison R Moliterno ◽  
Donna Marie Williams ◽  
Jonathan M. Gerber ◽  
Michael A McDevitt ◽  
Ophelia Rogers ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Single Cell ◽  
Board Of Directors ◽  
Disease Burden ◽  
Disease Duration ◽  
Single Cells ◽  
Advisory Committees ◽  
Allele Burden ◽  
Wide Range

Abstract Introduction: Essential thrombocytosis (ET), polycythemia vera (PV), and myelofibrosis (MF; post ETMF, post PVMF and primary MF) share the JAK2V617F mutation, but differ with regard to clinical phenotype, rate of disease progression, and risk of transformation. Variation in the JAK2V617F neutrophil allele burden does not account for these observed differences in clinical behavior or natural history. We therefore investigated the JAK2V617F burden and JAK2 genotype composition in the hematopoietic stem cell (HSC) population of MPN patients. Methods: We studied 47 JAK2V617F-positive MPN patients during 51 distinct disease phases. Circulating CD34+ cells were flow-sorted based on the stem cell markers CD34, CD38 and aldehyde dehydrogenase (ALDH). CD34+ CD38- ALDH+ HSC were sorted into 96 well plates and single cell JAK2 genotypes (average 40 single cells genotyped/patient with >1000 total single cells genotyped) were obtained using a nested PCR assay. Additional genomic lesions and chromosomal copy number variation were investigated in the sorted, single cell fractions in informative patients by FISH or multiplex single cell PCR. Distribution of JAK2V617F stem cell genotypes were correlated with disease phenotype, neutrophil JAK2V617F allele burden, splenomegaly, white cell count, chemotherapy requirement and disease evolution. Results: In all MPN cases, regardless of disease class, the JAK2V617F mutation was detected in the CD34+ CD38- ALDH+ fraction - the same population in which normal HSC reside. All ET and MF patients, and the majority of PV patients, had three JAK2 genotypes coexisting in their respective HSC populations. ET was characterized by a high percentage of JAK2WT stem cells (>75%) despite the concomitant presence of JAK2V617F homozygous clones and disease durations >15 years. Importantly, in the ET patients where JAK2WT clones fell to less than 50%, a PV phase followed. MF was characterized by a relatively low percentage of JAK2WT stem cells (median 24%), regardless of disease duration. PV had the most variable JAK2 genotypes with a wide range of JAK2WT stem cells (4%-92%) and a wide range of JAK2V617F homozygous stem cells (2-100%), and in 5/16 PV cases, only JAK2WT and JAK2V617F homozygous stem cells were identified. PV patients with JAK2V617F homozygous clones comprising more than 50% of their stem cells, regardless of disease duration, had higher white cell counts, higher neutrophil allele burdens, larger spleens and higher prevalence of chemotherapy compared to PV patients who had less than 50% JAK2V617F homozygous HSCs. The percentage of JAK2V617F homozygous HSC did not correlate with disease duration: some PV patients with a disease duration of >18 years had less than 10 % JAK2V617F homozygous HSC. A JAK2V617F - positive PV patient with a high JAK2V617F HSC burden and a high neutrophil JAK2V617F burden transformed to a JAK2V617F-negative chronic myelomonocytic leukemia (CMMoL); at the time of HSC analysis, the neutrophil JAK2V617F allele burden was 0% (previously 90%) and the HSC JAK2V617F homozygous percentage fell to 3% (previously 60%). While this patient's CMMoL was molecularly undefined, lesions identified in other JAK2V617F-positive patients (including mutations of ASXL1, TET2, deletion of 5q, 7q and 11q, trisomy 8 and 9), were also found in the CD34+ CD38- ALDH+ HSCs using single cell techniques, sometime coexistent with JAK2V617F-positive HSC, and sometimes in JAK2WT HSC. Conclusion: Driver and progression lesions in the JAK2V617F-positive MPN are acquired at the primitive HSC level. Despite decades of disease, the HSC pool in the MPN is mosaic for acquired lesions and also retains JAK2WT clones. Dominance of a particular JAK2 genotype at the primitive HSC level is variable, and distinguishes ET, where JAK2WT stem cells outnumber JAK2V617F-positive HSC, from MF, where JAK2WT HSC are the minority. PV is the most variable of the three MPN with regard to JAK2 genotype mosaicism. The allelic burden of HSC JAK2V617F in PV correlates with clinical disease burden. However, neither time nor JAK2V617F genotype determines the HSC burden in ET and PV, indicating that an undefined factor is a modifier of this important disease-defining process. Understanding the biology of HSC JAK2V617F homozygous clonal dominance may define an exploitable target to control disease burden, and to mitigate disease progression and evolution. Disclosures Moliterno: incyte: Membership on an entity's Board of Directors or advisory committees. Spivak:Incyte: Membership on an entity's Board of Directors or advisory committees.

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