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

Lareau, Caleb A.; Ma, Sai; Duarte, Fabiana M.; Buenrostro, Jason D.

doi:10.1038/s41467-020-14667-5

Cited by 48 publications

(28 citation statements)

References 20 publications

(27 reference statements)

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“…For all comparisons shown in the boxplots and violin plots, the top 1,000 cells/barcodes based on chromatin library complexity were plotted. The top 1,000 number was chosen to ensure the selection of real cells rather than barcode multiplets 61 or other barcodes associated with low counts. For the overall coverage comparison ( Fig.…”

Section: Methodsmentioning

confidence: 99%

Massively parallel single-cell mitochondrial DNA genotyping and chromatin profiling

et al. 2020

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Natural mitochondrial DNA (mtDNA) mutations enable the inference of clonal relationships among cells. mtDNA can be profiled along with measures of cell state, but has not yet been combined with the massively parallel approaches needed to tackle the complexity of human tissue. Here, we introduce a high-throughput, droplet-based mitochondrial single-cell Assay for Transposase Accessible Chromatin with sequencing (mtscATAC-seq), a method that combines high-confidence mtDNA mutation calling in thousands of single cells with their concomitant high-quality accessible chromatin profile. This enables the inference of mtDNA heteroplasmy, clonal relationships, cell state, and accessible chromatin variation in individual cells. We reveal single-cell variation in heteroplasmy of a pathologic mtDNA variant, which we associate with intra-individual chromatin variability and clonal evolution. We clonally trace thousands of cells from cancers, linking epigenomic variability to subclonal evolution and infer cellular dynamics of differentiating hematopoietic cells in vitro and in vivo . Taken together, our approach enables the study of cellular population dynamics and clonal properties in vivo .

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Section: Methodsmentioning

confidence: 99%

Massively parallel single-cell mitochondrial DNA genotyping and chromatin profiling

et al. 2020

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“…Another critical feature of DisCo is the use of machinevision to obtain full control of the entire co-encapsulation process including particle detection, particle positioning, particle droplet injection, and droplet volume. This enables the correct assembly of most droplets, virtually eradicating confounding factors that arise due to failed coencapsulations 37,38 . In concept, DisCo is thus fundamentally different to passive particle pairing approaches such as traps [39][40][41] and, compared to these technologies, offers the advantage of requiring vastly simpler and reusable chips without suffering from cell/particle size and shape selection biases 13,42 .…”

Section: Discussionmentioning

confidence: 99%

Deterministic scRNA-seq of individual intestinal organoids reveals new subtypes and coexisting distinct stem cell pools

Bues

Biočanin

Pezoldt

et al. 2020

Preprint

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1While Single-cell RNA-sequencing (scRNA-seq) has transformed our ability to resolve cellular 2 properties across systems, current microfluidics-based scRNA-seq technologies are limited to 3 samples with large amounts of cells (> 1,000 cells). This prevents efficient processing of 4 individual, small tissue samples, leading to confounded mosaic cell population read-outs. To 5 overcome these limitations, we developed a deterministic, mRNA-capture bead and cell co-6 encapsulation dropleting system, DisCo, that enables precise particle and cell positioning and 7 droplet sorting control through combined machine-vision and multilayer microfluidics. To 8 underscore the unique capabilities of our approach in processing low-input samples (< 100 cells) 9 at high efficiency (> 70%), we analyzed intestinal organoid development by "DisCo-ing" 31 10 individual organoids at varying developmental stages. This revealed extensive organoid 11 heterogeneity, uncovering a so far uncharacterized "gobloid" subtype consisting predominantly of 12 precursor and mature (Muc2 + ) goblet cells. We also identified spheroids that are comprised of a 13 secondary regenerative fetal-like Ly6a + stem cell population, marked by prolonged YAP1 nuclear 14 translocation and target gene expression, and that persist as symmetrical cysts even under 15 differentiation conditions. These findings illustrate the power of our "no-cell-left-behind" platform 16 in providing high-resolution snapshots of cellular heterogeneity among individual, small tissues. 17 18 19 20 21 22 23 24 25 26

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“…The goal of these analyses is to infer gene regulatory networks and identify their key regulators. Analytical tools specifically designed for scATAC-seq datasets either with or without scRNAseq, including chromeVAR, Signac, and MAESTRO, were developed, although computational challenges remain [ 87 , 88 , 89 , 90 , 91 ].…”

Section: Multimodal Profiling Of Hiv-1 Latencymentioning

confidence: 99%

Leveraging Novel Integrated Single-Cell Analyses to Define HIV-1 Latency Reversal

Zhao

Tsibris

2021

Viruses

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While suppressive antiretroviral therapy can effectively limit HIV-1 replication and evolution, it leaves behind a residual pool of integrated viral genomes that persist in a state of reversible nonproductive infection, referred to as the HIV-1 reservoir. HIV-1 infection models were established to investigate HIV-1 latency and its reversal; recent work began to probe the dynamics of HIV-1 latency reversal at single-cell resolution. Signals that establish HIV-1 latency and govern its reactivation are complex and may not be completely resolved at the cellular and regulatory levels by the aggregated measurements of bulk cellular-sequencing methods. High-throughput single-cell technologies that characterize and quantify changes to the epigenome, transcriptome, and proteome continue to rapidly evolve. Combinations of single-cell techniques, in conjunction with novel computational approaches to analyze these data, were developed and provide an opportunity to improve the resolution of the heterogeneity that may exist in HIV-1 reactivation. In this review, we summarize the published single-cell HIV-1 transcriptomic work and explore how cutting-edge advances in single-cell techniques and integrative data-analysis tools may be leveraged to define the mechanisms that control the reversal of HIV-1 latency.

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

Cited by 48 publications

References 20 publications

Massively parallel single-cell mitochondrial DNA genotyping and chromatin profiling

Massively parallel single-cell mitochondrial DNA genotyping and chromatin profiling

Deterministic scRNA-seq of individual intestinal organoids reveals new subtypes and coexisting distinct stem cell pools

Leveraging Novel Integrated Single-Cell Analyses to Define HIV-1 Latency Reversal

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