InDrops and Drop-seq technologies for single-cell sequencing

Klein, Allon M.; Macosko, Evan Z.

doi:10.1039/c7lc90070h

Cited by 42 publications

(30 citation statements)

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“…roplet-based partitioning systems have become an essential tool for single-cell genomics research. In contrast to platebased single-cell assays, droplet-based methods, including scRNA-seq 1,2 and scATAC-seq 3,4 enable profiling of thousands of cells in a single experiment. The marked increase in throughput is achieved by parallel barcoding of cellular nucleic acids with beads containing high-diversity DNA barcodes.…”

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confidence: 99%

Inference and effects of barcode multiplets in droplet-based single-cell assays

Lareau

Duarte

et al. 2020

Nat Commun

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A widespread assumption for single-cell analyses specifies that one cell's nucleic acids are predominantly captured by one oligonucleotide barcode. Here, we show that~13-21% of cell barcodes from the 10x Chromium scATAC-seq assay may have been derived from a droplet with more than one oligonucleotide sequence, which we call "barcode multiplets". We demonstrate that barcode multiplets can be derived from at least two different sources. First, we confirm that approximately 4% of droplets from the 10x platform may contain multiple beads. Additionally, we find that approximately 5% of beads may contain detectable levels of multiple oligonucleotide barcodes. We show that this artifact can confound single-cell analyses, including the interpretation of clonal diversity and proliferation of intra-tumor lymphocytes. Overall, our work provides a conceptual and computational framework to identify and assess the impacts of barcode multiplets in single-cell data.

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mentioning

confidence: 99%

Inference and effects of barcode multiplets in droplet-based single-cell assays

Lareau

Duarte

et al. 2020

Nat Commun

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“…While many single‐cell transcriptomics approaches can readily be applied in medium throughput on FACS‐sorted cells isolated into multi‐well plates, the scale and ease with which single‐cell transcriptomics experiments can be undertaken has benefited considerably from the introduction of combinatorial indexing, microfluidic and nanowell approaches to partition and/or barcode individual cells.…”

Section: Concepts and Methods For Single‐cell Isolation And Profilingmentioning

confidence: 99%

“…High‐throughput single‐cell gene expression profiling has been revolutionized by the introduction of microfluidics approaches which enable 3′ transcript counting from thousands of cells in parallel. The Drop‐seq and Indrop methods introduced this approach, in which individual cells or nuclei are co‐encapsulated in droplets with uniquely barcoded oligo‐dT primers, enabling cDNA to be pooled and sequenced in parallel, with reads assigned to individual cells based on their barcode. Droplet‐based methods have also been integrated with pooled CRISPR screening approaches to identify gene targets in thousands of single‐cells, using panels of guide RNAs …”

Section: Concepts and Methods For Single‐cell Isolation And Profilingmentioning

confidence: 99%

Defining Cell Identity with Single‐Cell Omics

et al. 2018

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Cells are a fundamental unit of life, and the ability to study the phenotypes and behaviors of individual cells is crucial to understanding the workings of complex biological systems. Cell phenotypes (epigenomic, transcriptomic, proteomic, and metabolomic) exhibit dramatic heterogeneity between and within the different cell types and states underlying cellular functional diversity. Cell genotypes can also display heterogeneity throughout an organism, in the form of somatic genetic variation—most notably in the emergence and evolution of tumors. Recent technical advances in single‐cell isolation and the development of omics approaches sensitive enough to reveal these aspects of cell identity have enabled a revolution in the study of multicellular systems. In this review, we discuss the technologies available to resolve the genomes, epigenomes, transcriptomes, proteomes, and metabolomes of single cells from a wide variety of living systems.

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“…2 With the emergence and maturation of droplet microfluidics in recent years, researchers have addressed the need for high throughput, low-cost assays by reducing reaction volumes. 3 Droplet microfluidics now allows for experiments on massive scales both within and outside of the "-omics" domain. 4,5 Focusing on single cell transcriptomics, the possibility to perform rapid compartmentalization has significantly increased the throughput and sensitivity of analyses at the single cell level.…”

Section: Introductionmentioning

confidence: 99%