High-Throughput Single-Cell RNA-Seq of Large Cells and Nuclei

2017 ◽  
Vol 37 (17) ◽  
pp. 12-13
Author(s):  
Jennifer Chew ◽  
Adam Bemis ◽  
Ronald Lebofsky ◽  
Anna Quinlan ◽  
Kelly Kaihara
Keyword(s):  
BMC Genomics ◽  
2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Tracy M. Yamawaki ◽  
Daniel R. Lu ◽  
Daniel C. Ellwanger ◽  
Dev Bhatt ◽  
Paolo Manzanillo ◽  
...  

Abstract Background Elucidation of immune populations with single-cell RNA-seq has greatly benefited the field of immunology by deepening the characterization of immune heterogeneity and leading to the discovery of new subtypes. However, single-cell methods inherently suffer from limitations in the recovery of complete transcriptomes due to the prevalence of cellular and transcriptional dropout events. This issue is often compounded by limited sample availability and limited prior knowledge of heterogeneity, which can confound data interpretation. Results Here, we systematically benchmarked seven high-throughput single-cell RNA-seq methods. We prepared 21 libraries under identical conditions of a defined mixture of two human and two murine lymphocyte cell lines, simulating heterogeneity across immune-cell types and cell sizes. We evaluated methods by their cell recovery rate, library efficiency, sensitivity, and ability to recover expression signatures for each cell type. We observed higher mRNA detection sensitivity with the 10x Genomics 5′ v1 and 3′ v3 methods. We demonstrate that these methods have fewer dropout events, which facilitates the identification of differentially-expressed genes and improves the concordance of single-cell profiles to immune bulk RNA-seq signatures. Conclusion Overall, our characterization of immune cell mixtures provides useful metrics, which can guide selection of a high-throughput single-cell RNA-seq method for profiling more complex immune-cell heterogeneity usually found in vivo.


2019 ◽  
Vol 4 (4) ◽  
pp. 683-692 ◽  
Author(s):  
Mariona Nadal-Ribelles ◽  
Saiful Islam ◽  
Wu Wei ◽  
Pablo Latorre ◽  
Michelle Nguyen ◽  
...  
Keyword(s):  

2020 ◽  
Author(s):  
Chi-Ming Kevin Li ◽  
Tracy M Yamawaki ◽  
Daniel R Lu ◽  
Daniel C Ellwanger ◽  
Dev Bhatt ◽  
...  

Abstract Background: Elucidation of immune populations with single-cell RNA-seq has greatly benefited the fieldof immunology by deepening the characterization of immune heterogeneity and leading to thediscovery of new subtypes. However, single-cell methods inherently suffer from limitations in therecovery of complete transcriptomes due to the prevalence of cellular and transcriptional dropoutevents. This issue is often compounded by limited sample availability and limited prior knowledge ofheterogeneity, which can confound data interpretation.Results: Here, we systematically benchmarked seven high-throughput single-cell RNA-seq methods. Weprepared 21 libraries under identical conditions of a defined mixture of two human and two murinelymphocyte cell lines, simulating heterogeneity across immune-cell types and cell sizes. We evaluatemethods by their cell recovery rate, library efficiency, sensitivity, and ability to recover expressionsignatures for each cell type. We observed higher mRNA detection sensitivity with the 10x Genomics 5’v1 and 3’ v3 methods. We demonstrate that these methods have fewer drop-out events whichfacilitates the identification of differentially-expressed genes and improves the concordance of singlecellprofiles to immune bulk RNA-seq signatures.Conclusion: Overall, our characterization of immune cell mixtures provides useful metrics, which canguide selection of a high-throughput single-cell RNA-seq method for profiling more complex immunecellheterogeneity usually found in vivo.


2018 ◽  
Vol 52 (1) ◽  
pp. 203-221 ◽  
Author(s):  
Kenneth D. Birnbaum

The growing scale and declining cost of single-cell RNA-sequencing (RNA-seq) now permit a repetition of cell sampling that increases the power to detect rare cell states, reconstruct developmental trajectories, and measure phenotype in new terms such as cellular variance. The characterization of anatomy and developmental dynamics has not had an equivalent breakthrough since groundbreaking advances in live fluorescent microscopy. The new resolution obtained by single-cell RNA-seq is a boon to genetics because the novel description of phenotype offers the opportunity to refine gene function and dissect pleiotropy. In addition, the recent pairing of high-throughput genetic perturbation with single-cell RNA-seq has made practical a scale of genetic screening not previously possible.


PLoS ONE ◽  
2015 ◽  
Vol 10 (5) ◽  
pp. e0116328 ◽  
Author(s):  
Assaf Rotem ◽  
Oren Ram ◽  
Noam Shoresh ◽  
Ralph A. Sperling ◽  
Michael Schnall-Levin ◽  
...  

2021 ◽  
Author(s):  
Ryan Dohn ◽  
Bingqing Xie ◽  
Rebecca Back ◽  
Alan Selewa ◽  
Heather Eckart ◽  
...  

Advances in high-throughput single-cell mRNA sequencing (scRNA-seq) have been limited till date by technical challenges like tough cell walls and low RNA quantity that prevented transcriptomic profiling of microbial species at throughput. We present microbial Drop-seq or mDrop-seq, a high-throughput scRNA-seq technique that is used on two yeast species, Saccharomyces cerevisiae, a popular model organism and Candida albicans, a common opportunistic pathogen. We benchmarked mDrop-seq for sensitivity and specificity and used it to profile 35,109 S. cerevisiae cells to detect variation in mRNA levels between them. As a proof of concept, we quantified expression differences in heat-shocked S. cerevisiae using mDrop-seq. We detected differential activation of stress response genes within a seemingly homogenous population of S. cerevisiae under heat-shock. We also applied mDrop-seq to C. albicans cells, a polymorphic and clinically relevant yeast species with thicker cell wall compared to S. cerevisiae. Single cell transcriptomes in 39,705 C. albicans cells was characterized using mDrop-seq under different conditions, including exposure to fluconazole, a common anti-fungal drug. We noted differential regulation in stress response and drug target pathways between C. albicans cells, changes in cell cycle patterns and marked increases in histone activity. These experiments are among the first high throughput single cell RNA-seq on different yeast species and demonstrate mDrop-seq as an affordable, easily implementable, and scalable technique that can quantify the variability in gene expression in different yeast species. We hope that mDrop-seq will lead to better understanding of genetic variation in yeasts in response to stimuli and find immediate applications in investigating drug resistance and infection outcome.


2021 ◽  
Vol 2021 ◽  
pp. 1-18
Author(s):  
Chaohua Mo ◽  
Le Xie ◽  
Chang Chen ◽  
Jie Ma ◽  
Yingxin Huang ◽  
...  

Background. CDC28 Protein Kinase Regulatory Subunit 1B (CKS1B) is a member of cyclin-dependent kinase subfamily and the relationship between CKS1B and osteosarcoma (OS) remains to be explored. Methods. 80 OS and 41 nontumor tissue samples were arranged to conduct immunohistochemistry (IHC) to evaluate CKS1B expression between OS and nontumor samples. The standard mean deviation (SMD) was calculated based on in-house IHC and tissue microarrays and exterior high-throughput datasets for further verification of CKS1B expression in OS. The effect of CKS1B expression on clinicopathological and overall survival of OS patients was measured through public high-throughput datasets, and analysis of immune infiltration and single-cell RNA-seq was applied to ascertain molecular mechanism of CKS1B in OS. Results. A total of 197 OS samples and 83 nontumor samples (including tissue and cell line) were obtained from in-house IHC, microarrays, and exterior high-throughput datasets. The analysis of integrated expression status demonstrated upregulation of CKS1B in OS (SMD = 1.38, 95% CI [0.52–2.25]) and the significant power of CKS1B expression in distinguishing OS samples from nontumor samples (Area under the Curve (AUC) = 0.89, 95% CI [0.86–0.91]). Clinicopathological and prognosis analysis indicated no remarkable significance but inference of immune infiltration and single-cell RNA-seq prompted that OS patients with overexpressed CKS1B were more likely to suffer OS metastasis while MYC Protooncogene may be the upstream regulon of CKS1B in proliferating osteoblastic OS cells. Conclusions. In this study, sufficient evidence was provided for upregulation of CKS1B in OS. The advanced effect of CKS1B on OS progression indicates a foreground of CKS1B as a biomarker for OS.


2021 ◽  
Author(s):  
Tyler E. Miller ◽  
Caleb A. Lareau ◽  
Julia A. Verga ◽  
Daniel Ssozi ◽  
Leif S. Ludwig ◽  
...  

AbstractReconstructing lineage relationships in complex tissues can reveal mechanisms underlying development and disease. Recent methods combine single-cell transcriptomics with mitochondrial DNA variant detection to establish lineage relationships in primary human cells, but are not scalable to interrogate complex tissues. To overcome this limitation, here we develop a technology for high-confidence detection of mitochondrial mutations from high-throughput single-cell RNA-sequencing. We use the new method to identify skewed immune cell expansions in primary human clonal hematopoiesis.


Author(s):  
Lili Blumenberg ◽  
Kelly V. Ruggles

AbstractUnsupervised clustering is a common and exceptionally useful tool for large biological datasets. However, clustering requires upfront algorithm and hyperparameter selection, which can introduce bias into the final clustering labels. It is therefore advisable to obtain a range of clustering results from multiple models and hyperparameters, which can be cumbersome and slow. To streamline this process, we present hypercluster, a python package and SnakeMake pipeline for flexible and parallelized clustering evaluation and selection. Hypercluster is available on bioconda; installation, documentation and example workflows can be found at: https://github.com/ruggleslab/hypercluster.Author summaryUnsupervised clustering is a technique for grouping similar samples within a dataset. It is extremely common when analyzing big data from patient samples, or high throughput techniques like single cell RNA-seq. When researchers use unsupervised clustering, they have to select parameters that affect the final result—for instance, how many groups they expect to find or what the smallest group is allowed to be. Some methods require setting even less intuitive parameters. For most applications, it is extremely challenging to guess what the values of these parameters should be; therefore to prevent introducing bias into the final results, researchers should test many different parameters and methods to find the best groups. This process is cumbersome, slow and challenging to perform in a reproducible way. We developed hypercluster, a tool that automates this process, make it much faster, and presenting the results in a reproducible and helpful manner.


2016 ◽  
Author(s):  
Weiqiang Zhou ◽  
Zhicheng Ji ◽  
Hongkai Ji

Conventional high-throughput technologies for mapping regulatory element activities such as ChIP-seq, DNase-seq and FAIRE-seq cannot analyze samples with small number of cells. The recently developed ATAC-seq allows regulome mapping in small-cell-number samples, but its signal in single cell or samples with ≤500 cells remains discrete or noisy. Compared to these technologies, measuring transcriptome by RNA-seq in single-cell and small-cell-number samples is more mature. Here we show that one can globally predict chromatin accessibility and infer regulome using RNA-seq. Genome-wide chromatin accessibility predicted by RNA-seq from 30 cells is comparable with ATAC-seq from 500 cells. Predictions based on single-cell RNA-seq can more accurately reconstruct bulk chromatin accessibility than using single-cell ATAC-seq by pooling the same number of cells. Integrating ATAC-seq with predictions from RNA-seq increases power of both methods. Thus, transcriptome-based prediction can provide a new tool for decoding gene regulatory programs in small-cell-number samples.


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