scholarly journals A comparison between single cell RNA sequencing and single molecule RNA FISH for rare cell analysis

2017 ◽  
Author(s):  
Eduardo Torre ◽  
Hannah Dueck ◽  
Sydney Shaffer ◽  
Janko Gospocic ◽  
Rohit Gupte ◽  
...  
Keyword(s):  
Single Cell ◽  
Rna Sequencing ◽  
Single Molecule ◽  
Single Cells ◽  
Cycle Phase ◽  
Expression Variability ◽  
Sequencing Technologies ◽  
Rna Fish ◽  
Single Cell Rna Sequencing ◽  
Sequencing Platforms

AbstractThe development of single cell RNA sequencing technologies has emerged as a powerful means of profiling the transcriptional behavior of single cells, leveraging the breadth of sequencing measurements to make inferences about cell type. However, there is still little understanding of how well these methods perform at measuring single cell variability for small sets of genes and what “transcriptome coverage” (e.g. genes detected per cell) is needed for accurate measurements. Here, we use single molecule RNA FISH measurements of 26 genes in thousands of melanoma cells to provide an independent reference dataset to assess the performance of the DropSeq and Fluidigm single cell RNA sequencing platforms. We quantified the Gini coefficient, a measure of rare-cell expression variability, and find that the correspondence between RNA FISH and single cell RNA sequencing for Gini, unlike for mean, increases markedly with per-cell library complexity up to a threshold of ∼2000 genes detected. A similar complexity threshold also allows for robust assignment of multi-genic cell states such as cell cycle phase. Our results provide guidelines for selecting sequencing depth and complexity thresholds for single cell RNA sequencing. More generally, our results suggest that if the number of genes whose expression levels are required to answer any given biological question is small, then greater transcriptome complexity per cell is likely more important than obtaining very large numbers of cells.

scholarly journals Rare Cell Detection by Single-Cell RNA Sequencing as Guided by Single-Molecule RNA FISH

Cell Systems ◽  
2018 ◽  
Vol 6 (2) ◽  
pp. 171-179.e5 ◽  
Author(s):  
Eduardo Torre ◽  
Hannah Dueck ◽  
Sydney Shaffer ◽  
Janko Gospocic ◽  
Rohit Gupte ◽  
...  
Keyword(s):  
Single Cell ◽  
Rna Sequencing ◽  
Single Molecule ◽  
Cell Detection ◽  
Rna Fish ◽  
Single Cell Rna Sequencing

scholarly journals Single-Cell RNA Sequencing of Batch Chlamydomonas Cultures Reveals Heterogeneity in their Diurnal Cycle Phase

2021 ◽  
Author(s):  
Feiyang Ma ◽  
Patrice A Salomé ◽  
Sabeeha S Merchant ◽  
Matteo Pellegrini
Keyword(s):  
Cell Wall ◽  
Single Cell ◽  
Rna Sequencing ◽  
Environmental Changes ◽  
Single Cells ◽  
Nitrogen Deficiency ◽  
Cycle Phase ◽  
Rna Seq ◽  
Single Cell Rna Sequencing ◽  
A Cell

Abstract The photosynthetic unicellular alga Chlamydomonas (Chlamydomonas reinhardtii) is a versatile reference for algal biology because of its ease of culture in the laboratory. Genomic and systems biology approaches have previously described transcriptome responses to environmental changes using bulk data, thus representing the average behavior from pools of cells. Here, we apply single-cell RNA sequencing (scRNA-seq) to probe the heterogeneity of Chlamydomonas cell populations under three environments and in two genotypes differing by the presence of a cell wall. First, we determined that RNA can be extracted from single algal cells with or without a cell wall, offering the possibility to sample natural algal communities. Second, scRNA-seq successfully separated single cells into non-overlapping cell clusters according to their growth conditions. Cells exposed to iron or nitrogen deficiency were easily distinguished despite a shared tendency to arrest photosynthesis and cell division to economize resources. Notably, these groups of cells recapitulated known patterns observed with bulk RNA-seq, but also revealed their inherent heterogeneity. A substantial source of variation between cells originated from their endogenous diurnal phase, although cultures were grown in constant light. We exploited this result to show that circadian iron responses may be conserved from algae to land plants. We document experimentally that bulk RNA-seq data represent an average of typically hidden heterogeneity in the population.

scholarly journals Single-Cell RNA Sequencing of Batch Chlamydomonas Cultures Reveals Heterogeneity in their Diurnal Cycle Phase

2020 ◽  
Author(s):  
Feiyang Ma ◽  
Patrice A. Salomé ◽  
Sabeeha S. Merchant ◽  
Matteo Pellegrini
Keyword(s):  
Cell Wall ◽  
Single Cell ◽  
Rna Sequencing ◽  
Diurnal Cycle ◽  
Single Cells ◽  
Nitrogen Deficiency ◽  
Cycle Phase ◽  
Rna Seq ◽  
Single Cell Rna Sequencing ◽  
A Cell

ABSTRACTThe photosynthetic unicellular alga Chlamydomonas (Chlamydomonas reinhardtii) is a versatile reference for algal biology because of the facility with which it can be cultured in the laboratory. Genomic and systems biology approaches have previously been used to describe how the transcriptome responds to environmental changes, but this analysis has been limited to bulk data, representing the average behavior from pools of cells. Here, we apply single-cell RNA sequencing (scRNA-seq) to probe the heterogeneity of Chlamydomonas cell populations under three environments and in two genotypes differing in the presence of a cell wall. First, we determined that RNA can be extracted from single algal cells with or without a cell wall, offering the possibility to sample algae communities in the wild. Second, scRNA-seq successfully separated single cells into non-overlapping cell clusters according to their growth conditions. Cells exposed to iron or nitrogen deficiency were easily distinguished despite a shared tendency to arrest cell division to economize resources. Notably, these groups of cells recapitulated known patterns observed with bulk RNA-seq, but also revealed their inherent heterogeneity. A substantial source of variation between cells originated from their endogenous diurnal phase, although cultures were grown in constant light. We exploited this result to show that circadian iron responses may be conserved from algae to land plants. We propose that bulk RNA-seq data represent an average of varied cell states that hides underappreciated heterogeneity.One-sentence summaryWe show that single-cell RNA-seq (scRNA-seq) can be applied to Chlamydomonas cultures to reveal the that heterogenity in bulk cultures is largely driven by diurnal cycle phasesThe author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantcell.org) is: Matteo Pellegrini ([email protected])

scholarly journals The Comparison of Two Single-cell Sequencing Platforms: BD Rhapsody and 10x Genomics Chromium

2020 ◽  
Vol 21 (8) ◽  
pp. 602-609
Author(s):  
Caixia Gao ◽  
Mingnan Zhang ◽  
Lei Chen
Keyword(s):  
Single Cell ◽  
Rna Sequencing ◽  
Biomedical Research ◽  
Genomic Dna ◽  
Single Cells ◽  
Single Cell Sequencing ◽  
Single Cell Rna Sequencing ◽  
Sequencing Platforms ◽  
Dna Profiles ◽  
Transcription Expression

The cell is the unit of life for all organisms, and all cells are certainly not the same. So the technology to generate transcription expression or genomic DNA profiles from single cells is crucial. Since its establishment in 2009, single-cell RNA sequencing (scRNA-seq) has emerged as a major driver of progress in biomedical research. During the last three years, several new single-cell sequencing platforms have emerged. Yet there are only a few systematic comparisons of the advantages and limitations of these commonly used platforms. Here we compare two single-cell sequencing platforms: BD Rhapsody and 10x Genomics Chromium, including their different mechanisms and some scRNA-seq results obtained with them.

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 Quality assessment of single-cell RNA sequencing data by coverage skewness analysis

2019 ◽  
Author(s):  
Imad Abugessaisa ◽  
Shuhei Noguchi ◽  
Melissa Cardon ◽  
Akira Hasegawa ◽  
Kazuhide Watanabe ◽  
...  
Keyword(s):  
Quality Assessment ◽  
Single Cell ◽  
Rna Sequencing ◽  
Single Cells ◽  
Assessment Method ◽  
Poor Quality ◽  
Sequencing Data ◽  
Single Cell Rna Sequencing ◽  
Gene Coverage ◽  
The Impact

AbstractAnalysis and interpretation of single-cell RNA-sequencing (scRNA-seq) experiments are compromised by the presence of poor quality cells. For meaningful analyses, such poor quality cells should be excluded to avoid biases and large variation. However, no clear guidelines exist. We introduce SkewC, a novel quality-assessment method to identify poor quality single-cells in scRNA-seq experiments. The method is based on the assessment of gene coverage for each single cell and its skewness as a quality measure. To validate the method, we investigated the impact of poor quality cells on downstream analyses and compared biological differences between typical and poor quality cells. Moreover, we measured the ratio of intergenic expression, suggesting genomic contamination, and foreign organism contamination of single-cell samples. SkewC is tested in 37,993 single-cells generated by 15 scRNA-seq protocols. We envision SkewC as an indispensable QC method to be incorporated into scRNA-seq experiment to preclude the possibility of scRNA-seq data misinterpretation.

scholarly journals Assessing the measurement transfer function of single-cell RNA sequencing

2016 ◽  
Author(s):  
Hannah R. Dueck ◽  
Rizi Ai ◽  
Adrian Camarena ◽  
Bo Ding ◽  
Reymundo Dominguez ◽  
...  
Keyword(s):  
Transfer Function ◽  
Single Cell ◽  
Rna Sequencing ◽  
Large Scale ◽  
Transfer Functions ◽  
Single Cells ◽  
Biological Information ◽  
Gene Detection ◽  
Single Cell Rna Sequencing ◽  
Scale Control

AbstractRecently, measurement of RNA at single cell resolution has yielded surprising insights. Methods for single-cell RNA sequencing (scRNA-seq) have received considerable attention, but the broad reliability of single cell methods and the factors governing their performance are still poorly known. Here, we conducted a large-scale control experiment to assess the transfer function of three scRNA-seq methods and factors modulating the function. All three methods detected greater than 70% of the expected number of genes and had a 50% probability of detecting genes with abundance greater than 2 to 4 molecules. Despite the small number of molecules, sequencing depth significantly affected gene detection. While biases in detection and quantification were qualitatively similar across methods, the degree of bias differed, consistent with differences in molecular protocol. Measurement reliability increased with expression level for all methods and we conservatively estimate the measurement transfer functions to be linear above ~5-10 molecules. Based on these extensive control studies, we propose that RNA-seq of single cells has come of age, yielding quantitative biological information.

scholarly journals Rheostat Coordination of Latent Kaposi Sarcoma-Associated Herpesvirus RNA expression in Single Cells

2021 ◽  
Author(s):  
Nicole C. Rondeau ◽  
JJ L. Miranda
Keyword(s):  
Single Cell ◽  
Rna Sequencing ◽  
Single Cells ◽  
Kaposi Sarcoma ◽  
Rna Expression ◽  
Single Cell Rna Sequencing ◽  
Rna Levels

We detected precise coordination of RNA levels between two latent genes of the Kaposi sarcoma-associated herpesvirus (KSHV) using single-cell RNA sequencing. LANA and vIL6 are expressed during latency by different promoters on remote regions of the episome.…

scholarly journals A spatially restricted fibrotic niche in pulmonary fibrosis is sustained by M-CSF/M-CSFR signalling in monocyte-derived alveolar macrophages

2019 ◽  
Vol 55 (1) ◽  
pp. 1900646 ◽  
Author(s):  
Nikita Joshi ◽  
Satoshi Watanabe ◽  
Rohan Verma ◽  
Renea P. Jablonski ◽  
Ching-I Chen ◽  
...  
Keyword(s):  
Pulmonary Fibrosis ◽  
Single Cell ◽  
Rna Sequencing ◽  
Single Molecule ◽  
Alveolar Macrophages ◽  
Lineage Tracing ◽  
Single Cell Rna Sequencing ◽  
Pharmacological Blockade ◽  
Macrophage Colony ◽  
Druggable Targets

Ontologically distinct populations of macrophages differentially contribute to organ fibrosis through unknown mechanisms.We applied lineage tracing, single-cell RNA sequencing and single-molecule fluorescence in situ hybridisation to a spatially restricted model of asbestos-induced pulmonary fibrosis.We demonstrate that tissue-resident alveolar macrophages, tissue-resident peribronchial and perivascular interstitial macrophages, and monocyte-derived alveolar macrophages are present in the fibrotic niche. Deletion of monocyte-derived alveolar macrophages but not tissue-resident alveolar macrophages ameliorated asbestos-induced lung fibrosis. Monocyte-derived alveolar macrophages were specifically localised to fibrotic regions in the proximity of fibroblasts where they expressed molecules known to drive fibroblast proliferation, including platelet-derived growth factor subunit A. Using single-cell RNA sequencing and spatial transcriptomics in both humans and mice, we identified macrophage colony-stimulating factor receptor (M-CSFR) signalling as one of the novel druggable targets controlling self-maintenance and persistence of these pathogenic monocyte-derived alveolar macrophages. Pharmacological blockade of M-CSFR signalling led to the disappearance of monocyte-derived alveolar macrophages and ameliorated fibrosis.Our findings suggest that inhibition of M-CSFR signalling during fibrosis disrupts an essential fibrotic niche that includes monocyte-derived alveolar macrophages and fibroblasts during asbestos-induced fibrosis.

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