Simultaneous multiplexed amplicon sequencing and transcriptome profiling in single cells

Saikia, Mridusmita; Burnham, Philip; Keshavjee, S.; Wang, Michael F. Z.; Heyang, Michael; Moral-Lopez, Pablo; Hinchman, Meleana M.; Danko, Charles G.; Parker, John S. L.; Vlaminck, Iwijn De

doi:10.1101/328328

Cited by 13 publications

(14 citation statements)

References 40 publications

(185 reference statements)

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“…Newer 3′-based methods that allow for higher throughput, and greater sample sizes at reasonable cost, have allowed the identification of rarer populations, such as ε-cells ( 84 ). Targeted sequencing approaches, such as droplet-assisted RNA targeting by single-cell sequencing (DART-Seq), significantly improve coverage by targeting the limited depth of scRNA-Seq to a subset of preselected transcripts of interest ( 85 ). Computational methods are being developed to take into account and correct for confounding factors, such as donor genetic variation, dropout, and technical noise, although avoiding confounders will always be preferable to correcting for them through bioinformatic means ( 86 , 87 ).…”

Section: Future Outlookmentioning

confidence: 99%

Navigating the Depths and Avoiding the Shallows of Pancreatic Islet Cell Transcriptomes

Mawla

Huising

2019

Diabetes

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Islet gene expression has been widely studied to better understand the transcriptional features that define a healthy β-cell. Transcriptomes of FACS-purified α-, β-, and δ-cells using bulk RNA-sequencing have facilitated our understanding of the complex network of cross talk between islet cells and its effects on β-cell function. However, these approaches were by design not intended to resolve heterogeneity between individual cells. Several recent studies used single-cell RNA sequencing (scRNA-Seq) to report considerable heterogeneity within mouse and human β-cells. In this Perspective, we assess how this newfound ability to assess gene expression at single-cell resolution has enhanced our understanding of β-cell heterogeneity. We conduct a comprehensive assessment of several single human β-cell transcriptome data sets and ask if the heterogeneity reported by these studies showed overlap and concurred with previously known examples of β-cell heterogeneity. We also illustrate the impact of the inevitable limitations of working at or below the limit of detection of gene expression at single cell resolution and their consequences for the quality of single–islet cell transcriptome data. Finally, we offer some guidance on when to opt for scRNA-Seq and when bulk sequencing approaches may be better suited.

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Section: Future Outlookmentioning

confidence: 99%

Navigating the Depths and Avoiding the Shallows of Pancreatic Islet Cell Transcriptomes

Mawla

Huising

2019

Diabetes

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“…By synthesizing target sequence-linked beads, targeted sequences could be captured directly without laborious PCR amplification of each targeted sequence (32). Alternatively, ligating target sequences to conventional beads could also be used (33). Another application involves coupling of methods targeting individual cells using barcoding (34).…”

Section: Discussionmentioning

confidence: 99%

Single-cell analysis of a mutant library generated using CRISPR-guided deaminase

Jun

Lim

Chun³

et al. 2019

Preprint

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CRISPR-based screening methods using single-cell RNA sequencing (scRNA-seq) technology enable comprehensive profiling of gene perturbations from knock-out mutations. However, evaluating substitution mutations using scRNA-seq is currently limited. We combined CRISPR RNAguided deaminase and scRNA-seq technology to develop a platform for introducing mutations in multiple genes and assessing the mutationassociated signatures. Using this platform, we generated a library consisting of 420 sgRNAs, performed sgRNA tracking analysis, and assessed the effect size of the response to vemurafenib in the melanoma cell line, which has been well-studied via knockout-based drop-out screens. However, a substitution mutation library screen has not been applied and transcriptional information for mechanisms of action was not assessed. Our platform permits discrimination of several candidate mutations that function differently from other mutations by integrating sgRNA candidates and gene expression readout. We anticipate that our platform will enable high-throughput analyses of the mechanisms related to a variety of biological events.

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“…For example, single-cell profiling of BCR repertoires and transcriptomes has recently revealed a high degree of bystander activation during influenza vaccination 36 . This combined immune repertoire and transcriptome profiling has also been employed to obtain unprecedented resolution of lymphocyte dynamics in the context of cancer 41,42 .…”

Section: Introductionmentioning

confidence: 99%

Single-cell immune repertoire and transcriptome sequencing reveals that clonally expanded and transcriptionally distinct lymphocytes populate the aged central nervous system in mice

Yermanos

Neumeier

Sandu

et al. 2020

Preprint

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One Sentence SummaryClonally expanded and transcriptionally distinct B and T cells are discovered in brains of elderly mice by single-cell immune repertoire and transcriptome sequencing. AbstractNeuroinflammation plays a crucial role during ageing and various neurological conditions, including Alzheimer's disease, multiple sclerosis and infection. Technical limitations, however, have prevented an integrative analysis of how lymphocyte immune receptor repertoires and their accompanying transcriptional states change with age in the central nervous system (CNS). Here, we leveraged single-cell sequencing to simultaneously profile B cell receptor (BCR) and T cell receptor (TCR) repertoires and accompanying gene expression profiles in young and old mouse brains. We observed the presence of clonally expanded B and T cells in the central nervous system (CNS) of aged mice. Furthermore, many of these B cells were of the IgM and IgD isotype and had low levels of somatic hypermutation. Integrating gene expression information additionally revealed distinct transcriptional profiles of these clonally expanded lymphocytes. Our findings implicate that clonally related T and B cells in the CNS of elderly mice may contribute to neuroinflammation accompanying homeostatic ageing.

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Simultaneous multiplexed amplicon sequencing and transcriptome profiling in single cells

Cited by 13 publications

References 40 publications

Navigating the Depths and Avoiding the Shallows of Pancreatic Islet Cell Transcriptomes

Navigating the Depths and Avoiding the Shallows of Pancreatic Islet Cell Transcriptomes

Single-cell analysis of a mutant library generated using CRISPR-guided deaminase

Single-cell immune repertoire and transcriptome sequencing reveals that clonally expanded and transcriptionally distinct lymphocytes populate the aged central nervous system in mice

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