Single-Cell Genomics: Approaches and Utility in Immunology

Neu, Karlynn E.; Tang, Qingming; Wilson, Patrick C.; Khan, Aly A.

doi:10.1016/j.it.2016.12.001

Cited by 62 publications

(57 citation statements)

References 67 publications

(103 reference statements)

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“…Single cell technology is a powerful tool for studying the growth, physiology, function and biodiversity of microorganisms, especially for the as-yet-unculturable microorganisms in nature (1)(2)(3)(4)(5). As the basis and most important step of a single cell study, single cell isolation has been applied in many research fields such as single cell genomics (6), neurobiology (7) and analysis of disease processes (8).…”

Section: Introductionmentioning

confidence: 99%

Laser induced isolation and cultivation of single microbial cells

Peng

Wang

Wang³

et al. 2020

Preprint

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Single cell isolation and cultivation play an important role in studying physiology, gene expression and functions of microorganisms. Laser Induced Forward Transfer Technique (LIFT) has been applied to isolate single cells but the cell viability after sorting is unclear. We demonstrate that a three-layer LIFT system could be applied to isolate single cells of Gram-negative (E. coli), Gram-positive (Lactobacillus rhamnosus GG, LGG), and eukaryotic microorganisms (Saccharomyces cerevisiae) and the sorted single cells were able to be cultured. The experiment results showed that the average cultivation recovery rate of the ejected single cells were 58% for Saccharomyces cerevisiae, 22% for E. coli, and 74% for Lactobacillus rhamnosus GG (LGG). The identities of the cultured cells from single cell sorting were confirmed by using colony PCR with 16S-rRNA for bacteria and large unit rRNA for yeast and subsequent sequencing. This precise sorting and cultivation technique of live single microbial cells can be coupled with other microscopic approaches (e.g. fluorescent and Raman microscopy) to culture single microorganisms with specific functions, revealing their roles in the natural community.ImportanceSingle cell isolation and cultivation are crucial to recover microorganisms for the study of physiology, gene expression and functions. We developed a laser induced cell sorting technology to precisely isolate single microbial cells from a microscopic slide. More importantly, the isolated single microbial cells are still viable for cultivation. We demonstrate to apply the live sorting method to isolate and cultivate single cells of Gram-negative (E. coli), Gram-positive (Lactobacillus rhamnosus GG, LGG), and eukaryotic microorganisms (Saccharomyces cerevisiae). This precise sorting and cultivation technique can be coupled with other microscopic approaches (e.g. fluorescent and Raman microscopy) to culture specifically targeted single microorganisms from microbial community.Abstract Graphic

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Section: Introductionmentioning

confidence: 99%

Laser induced isolation and cultivation of single microbial cells

Peng

Wang

Wang³

et al. 2020

Preprint

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“…Not all the known cells of the innate and adaptive immunity of hematopoietic origins were touched on, as our concern was to attempt to summarize the required components for immune microenvironment recapitulation. Besides, with the advent of single‐cell genomics, rare immune cell populations and new functions of known immune cells are emerging constantly . The immune niche is perhaps the personification of complexity, as the stromal cellular components are not passive in immunity, but indeed have active roles in recruiting immune cells, modulating immune response, and secreting stroma proteins; all this is achieved through the secretion of ECM components, chemokines, cytokines, and growth factors.…”

Section: Conclusion and Perspectives For The Futurementioning

confidence: 99%

Deconstructing Immune Microenvironments of Lymphoid Tissues for Reverse Engineering

Witzel¹,

Nasser

Garcia-Sabaté

et al. 2018

Adv Healthcare Materials

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The immune microenvironment presents a diverse panel of cues that impacts immune cell migration, organization, differentiation, and the immune response. Uniquely, both the liquid and solid phases of every specific immune niche within the body play an important role in defining cellular functions in immunity at that particular location. The in vivo immune microenvironment consists of biomechanical and biochemical signals including their gradients, surface topography, dimensionality, modes of ligand presentation, and cell–cell interactions, and the ability to recreate these immune biointerfaces in vitro can provide valuable insights into the immune system. This manuscript reviews the critical roles played by different immune cells and surveys the current progress of model systems for reverse engineering of immune microenvironments with a focus on lymphoid tissues.

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“…During the sequencing process, reverse transcription is necessary to convert RNA to cDNA for use in amplification, but this introduces positional bias regardless of where the polymerization begins (Hebenstreit, 2012). The following amplification process counteracts low input material, though this in turn leads to additional bias as some genes may experience preferential amplification, leading to uneven representation in the data (Neu et al, 2017). The amplification process also runs the risk of producing dropout events, in which either genes known to be present in a sample are completely absent from the observed gene counts or genes are observed with lower value than their true expression (Van den Berge et al, 2018).…”

Section: Introductionmentioning

confidence: 99%

Normalization Methods on Single-Cell RNA-seq Data: An Empirical Survey

Lytal

2020

Front. Genet.

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Data normalization is vital to single-cell sequencing, addressing limitations presented by low input material and various forms of bias or noise present in the sequencing process. Several such normalization methods exist, some of which rely on spike-in genes, molecules added in known quantities to serve as a basis for a normalization model. Depending on available information and the type of data, some methods may express certain advantages over others. We compare the effectiveness of seven available normalization methods designed specifically for single-cell sequencing using two real data sets containing spike-in genes and one simulation study. Additionally, we test those methods not dependent on spike-in genes using a real data set with three distinct cellcycle states and a real data set under the 10X Genomics GemCode platform with multiple cell types represented. We demonstrate the differences in effectiveness for the featured methods using visualization and classification assessment and conclude which methods are preferable for normalizing a certain type of data for further downstream analysis, such as classification or differential analysis. The comparison in computational time for all methods is addressed as well.

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Single-Cell Genomics: Approaches and Utility in Immunology

Cited by 62 publications

References 67 publications

Laser induced isolation and cultivation of single microbial cells

Laser induced isolation and cultivation of single microbial cells

Deconstructing Immune Microenvironments of Lymphoid Tissues for Reverse Engineering

Normalization Methods on Single-Cell RNA-seq Data: An Empirical Survey

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