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1.
Cell ; 179(5): 1207-1221.e22, 2019 Nov 14.
Artigo em Inglês | MEDLINE | ID: mdl-31730858

RESUMO

Accurate measurement of clonal genotypes, mutational processes, and replication states from individual tumor-cell genomes will facilitate improved understanding of tumor evolution. We have developed DLP+, a scalable single-cell whole-genome sequencing platform implemented using commodity instruments, image-based object recognition, and open source computational methods. Using DLP+, we have generated a resource of 51,926 single-cell genomes and matched cell images from diverse cell types including cell lines, xenografts, and diagnostic samples with limited material. From this resource we have defined variation in mitotic mis-segregation rates across tissue types and genotypes. Analysis of matched genomic and image measurements revealed correlations between cellular morphology and genome ploidy states. Aggregation of cells sharing copy number profiles allowed for calculation of single-nucleotide resolution clonal genotypes and inference of clonal phylogenies and avoided the limitations of bulk deconvolution. Finally, joint analysis over the above features defined clone-specific chromosomal aneuploidy in polyclonal populations.


Assuntos
Replicação do DNA/genética , Genoma Humano , Sequenciamento de Nucleotídeos em Larga Escala , Análise de Célula Única , Aneuploidia , Animais , Ciclo Celular/genética , Linhagem Celular Tumoral , Forma Celular , Sobrevivência Celular , Cromossomos Humanos/genética , Células Clonais , Elementos de DNA Transponíveis/genética , Diploide , Feminino , Genótipo , Humanos , Masculino , Camundongos , Mutação/genética , Filogenia , Polimorfismo de Nucleotídeo Único/genética
2.
Nat Methods ; 14(2): 167-173, 2017 02.
Artigo em Inglês | MEDLINE | ID: mdl-28068316

RESUMO

Single-cell genomics is critical for understanding cellular heterogeneity in cancer, but existing library preparation methods are expensive, require sample preamplification and introduce coverage bias. Here we describe direct library preparation (DLP), a robust, scalable, and high-fidelity method that uses nanoliter-volume transposition reactions for single-cell whole-genome library preparation without preamplification. We examined 782 cells from cell lines and triple-negative breast xenograft tumors. Low-depth sequencing, compared with existing methods, revealed greater coverage uniformity and more reliable detection of copy-number alterations. Using phylogenetic analysis, we found minor xenograft subpopulations that were undetectable by bulk sequencing, as well as dynamic clonal expansion and diversification between passages. Merging single-cell genomes in silico, we generated 'bulk-equivalent' genomes with high depth and uniform coverage. Thus, low-depth sequencing of DLP libraries may provide an attractive replacement for conventional bulk sequencing methods, permitting analysis of copy number at the cell level and of other genomic variants at the population level.


Assuntos
Genômica/métodos , Análise de Célula Única/métodos , Animais , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Feminino , Biblioteca Gênica , Humanos , Dispositivos Lab-On-A-Chip , Camundongos SCID , Filogenia , Análise de Célula Única/instrumentação , Ensaios Antitumorais Modelo de Xenoenxerto
3.
Proc Natl Acad Sci U S A ; 109(20): 7665-70, 2012 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-22547789

RESUMO

We present a programmable droplet-based microfluidic device that combines the reconfigurable flow-routing capabilities of integrated microvalve technology with the sample compartmentalization and dispersion-free transport that is inherent to droplets. The device allows for the execution of user-defined multistep reaction protocols in 95 individually addressable nanoliter-volume storage chambers by consecutively merging programmable sequences of picoliter-volume droplets containing reagents or cells. This functionality is enabled by "flow-controlled wetting," a droplet docking and merging mechanism that exploits the physics of droplet flow through a channel to control the precise location of droplet wetting. The device also allows for automated cross-contamination-free recovery of reaction products from individual chambers into standard microfuge tubes for downstream analysis. The combined features of programmability, addressability, and selective recovery provide a general hardware platform that can be reprogrammed for multiple applications. We demonstrate this versatility by implementing multiple single-cell experiment types with this device: bacterial cell sorting and cultivation, taxonomic gene identification, and high-throughput single-cell whole genome amplification and sequencing using common laboratory strains. Finally, we apply the device to genome analysis of single cells and microbial consortia from diverse environmental samples including a marine enrichment culture, deep-sea sediments, and the human oral cavity. The resulting datasets capture genotypic properties of individual cells and illuminate known and potentially unique partnerships between microbial community members.


Assuntos
Hidrodinâmica , Metagenoma/genética , Técnicas Analíticas Microfluídicas/instrumentação , Técnicas Analíticas Microfluídicas/métodos , Sequência de Bases , Primers do DNA/genética , Genótipo , Sedimentos Geológicos/microbiologia , Humanos , Processamento de Imagem Assistida por Computador , Metagenômica/métodos , Microscopia de Fluorescência , Dados de Sequência Molecular , Boca/microbiologia , Reação em Cadeia da Polimerase , RNA Ribossômico 16S/genética , Análise de Sequência de DNA , Tensoativos , Molhabilidade
4.
Genome Biol ; 20(1): 54, 2019 03 12.
Artigo em Inglês | MEDLINE | ID: mdl-30866997

RESUMO

Measuring gene expression of tumor clones at single-cell resolution links functional consequences to somatic alterations. Without scalable methods to simultaneously assay DNA and RNA from the same single cell, parallel single-cell DNA and RNA measurements from independent cell populations must be mapped for genome-transcriptome association. We present clonealign, which assigns gene expression states to cancer clones using single-cell RNA and DNA sequencing independently sampled from a heterogeneous population. We apply clonealign to triple-negative breast cancer patient-derived xenografts and high-grade serous ovarian cancer cell lines and discover clone-specific dysregulated biological pathways not visible using either sequencing method alone.


Assuntos
Biomarcadores Tumorais/genética , Cistadenocarcinoma Seroso/genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Modelos Estatísticos , Neoplasias Ovarianas/genética , Análise de Célula Única/métodos , Software , Neoplasias de Mama Triplo Negativas/genética , Animais , Células Clonais , Cistadenocarcinoma Seroso/patologia , Feminino , Humanos , Camundongos Endogâmicos NOD , Camundongos SCID , Neoplasias Ovarianas/patologia , Neoplasias de Mama Triplo Negativas/patologia , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de Xenoenxerto
5.
PLoS One ; 13(1): e0191601, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29377915

RESUMO

We present a microfluidic device for rapid gene expression profiling in single cells using multiplexed quantitative polymerase chain reaction (qPCR). This device integrates all processing steps, including cell isolation and lysis, complementary DNA synthesis, pre-amplification, sample splitting, and measurement in twenty separate qPCR reactions. Each of these steps is performed in parallel on up to 200 single cells per run. Experiments performed on dilutions of purified RNA establish assay linearity over a dynamic range of at least 104, a qPCR precision of 15%, and detection sensitivity down to a single cDNA molecule. We demonstrate the application of our device for rapid profiling of microRNA expression in single cells. Measurements performed on a panel of twenty miRNAs in two types of cells revealed clear cell-to-cell heterogeneity, with evidence of spontaneous differentiation manifested as distinct expression signatures. Highly multiplexed microfluidic RT-qPCR fills a gap in current capabilities for single-cell analysis, providing a rapid and cost-effective approach for profiling panels of marker genes, thereby complementing single-cell genomics methods that are best suited for global analysis and discovery. We expect this approach to enable new studies requiring fast, cost-effective, and precise measurements across hundreds of single cells.


Assuntos
Reação em Cadeia da Polimerase Multiplex/métodos , Limite de Detecção , Microfluídica/instrumentação
6.
Biotechnol J ; 10(10): 1546-54, 2015 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-26059045

RESUMO

Heterogeneity in the clonal outputs of individual human embryonic stem cells (hESCs) confounds analysis of their properties in studies of bulk populations and how to manipulate them for clinical applications. To circumvent this problem we developed a microfluidic device that supports the robust generation of colonies derived from single ESCs. This microfluidic system contains 160 individually addressable chambers equipped for perfusion culture of individual hESCs that could be shown to match the growth rates, marker expression and colony morphologies obtained in conventional cultures. Use of this microfluidic device to analyze the clonal growth kinetics of multiple individual hESCs induced to differentiation revealed variable shifts in the growth rate, area per cell and expression of OCT4 in the progeny of individual hESCs. Interestingly, low OCT4 expression, a slower growth rate and low nuclear to cytoplasmic ratios were found to be correlated responses. This study demonstrates how microfluidic systems can be used to enable large scale live-cell imaging of isolated hESCs exposed to changing culture conditions, to examine how different aspects of their variable responses are correlated.


Assuntos
Técnicas de Cultura de Células/métodos , Células-Tronco Embrionárias Humanas/citologia , Técnicas Analíticas Microfluídicas/métodos , Células-Tronco Pluripotentes/citologia , Técnicas de Cultura de Células/instrumentação , Diferenciação Celular/genética , Linhagem da Célula , Proliferação de Células/genética , Citometria de Fluxo , Heterogeneidade Genética , Humanos , Técnicas Analíticas Microfluídicas/instrumentação
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