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1.
BMC Genomics ; 21(1): 456, 2020 Jul 02.
Artigo em Inglês | MEDLINE | ID: mdl-32616006

RESUMO

BACKGROUND: The increasing demand of single-cell RNA-sequencing (scRNA-seq) experiments, such as the number of experiments and cells queried per experiment, necessitates higher sequencing depth coupled to high data quality. New high-throughput sequencers, such as the Illumina NovaSeq 6000, enables this demand to be filled in a cost-effective manner. However, current scRNA-seq library designs present compatibility challenges with newer sequencing technologies, such as index-hopping, and their ability to generate high quality data has yet to be systematically evaluated. RESULTS: Here, we engineered a dual-indexed library structure, called TruDrop, on top of the inDrop scRNA-seq platform to solve these compatibility challenges, such that TruDrop libraries and standard Illumina libraries can be sequenced alongside each other on the NovaSeq. On scRNA-seq libraries, we implemented a previously-documented countermeasure to the well-described problem of index-hopping, demonstrated significant improvements in base-calling accuracy on the NovaSeq, and provided an example of multiplexing twenty-four scRNA-seq libraries simultaneously. We showed favorable comparisons in transcriptional diversity of TruDrop compared with prior inDrop libraries. CONCLUSIONS: Our approach enables cost-effective, high throughput generation of sequencing data with high quality, which should enable more routine use of scRNA-seq technologies.


Assuntos
Sequenciamento de Nucleotídeos em Larga Escala , Análise de Sequência de RNA/métodos , Análise de Célula Única/métodos , Animais , Humanos , Camundongos , Alinhamento de Sequência , Análise de Sequência de RNA/normas , Análise de Célula Única/normas
3.
Nat Biotechnol ; 38(6): 715-721, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32231335

RESUMO

Mining the antibody repertoire of plasma cells and plasmablasts could enable the discovery of useful antibodies for therapeutic or research purposes1. We present a method for high-throughput, single-cell screening of IgG-secreting primary cells to characterize antibody binding to soluble and membrane-bound antigens. CelliGO is a droplet microfluidics system that combines high-throughput screening for IgG activity, using fluorescence-based in-droplet single-cell bioassays2, with sequencing of paired antibody V genes, using in-droplet single-cell barcoded reverse transcription. We analyzed IgG repertoire diversity, clonal expansion and somatic hypermutation in cells from mice immunized with a vaccine target, a multifunctional enzyme or a membrane-bound cancer target. Immunization with these antigens yielded 100-1,000 IgG sequences per mouse. We generated 77 recombinant antibodies from the identified sequences and found that 93% recognized the soluble antigen and 14% the membrane antigen. The platform also allowed recovery of ~450-900 IgG sequences from ~2,200 IgG-secreting activated human memory B cells, activated ex vivo, demonstrating its versatility.


Assuntos
Anticorpos/genética , Sequenciamento de Nucleotídeos em Larga Escala , Técnicas Analíticas Microfluídicas/instrumentação , Análise de Célula Única , Animais , Antígenos/imunologia , Linfócitos B/imunologia , Vacinas Anticâncer/imunologia , DNA/análise , DNA/genética , Sequenciamento de Nucleotídeos em Larga Escala/instrumentação , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Imunoglobulina G/genética , Camundongos , Análise de Célula Única/instrumentação , Análise de Célula Única/métodos
4.
Nat Genet ; 51(6): 1060-1066, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31152164

RESUMO

Modulation of chromatin structure via histone modification is a major epigenetic mechanism and regulator of gene expression. However, the contribution of chromatin features to tumor heterogeneity and evolution remains unknown. Here we describe a high-throughput droplet microfluidics platform to profile chromatin landscapes of thousands of cells at single-cell resolution. Using patient-derived xenograft models of acquired resistance to chemotherapy and targeted therapy in breast cancer, we found that a subset of cells within untreated drug-sensitive tumors share a common chromatin signature with resistant cells, undetectable using bulk approaches. These cells, and cells from the resistant tumors, have lost chromatin marks-H3K27me3, which is associated with stable transcriptional repression-for genes known to promote resistance to treatment. This single-cell chromatin immunoprecipitation followed by sequencing approach paves the way to study the role of chromatin heterogeneity, not just in cancer but in other diseases and healthy systems, notably during cellular differentiation and development.


Assuntos
Neoplasias da Mama/genética , Imunoprecipitação da Cromatina , Cromatina/genética , Heterogeneidade Genética , Sequenciamento de Nucleotídeos em Larga Escala , Análise de Célula Única , Cromatina/metabolismo , Biologia Computacional/métodos , Epigênese Genética , Feminino , Histonas/metabolismo , Humanos , Técnicas Analíticas Microfluídicas , Análise de Célula Única/métodos , Células Estromais , Fluxo de Trabalho
8.
IEEE Pulse ; 5(4): 4, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25158334
9.
IEEE Pulse ; 5(2): 4, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24757756
12.
Biotechniques ; 46(6): ii-viii, 2009 May.
Artigo em Inglês | MEDLINE | ID: mdl-19480642

RESUMO

Biomarkers discovered from gene expression profiles using hybridization microarrays have made great inroads in the diagnosis and development of safer and efficacious drugs. The candidate gene set is biologically validated by quantitative measurement with reverse transcriptase quantitative PCR (RT-qPCR) and is an effective strategy when implemented with microplates if the number of candidate genes and samples is small. With the trend toward informative candidate gene panels increasing from tens to hundreds of genes and sample cohorts exceeding several hundred, an alternative fluidic approach is needed that preserves the intrinsic analytical precision, large dynamic range, and high sensitivity of RT-qPCR, yet is scalable to high throughputs. We have evaluated the performance of a nanoliter fluidic system that enables up to 3072 nanoliter RT-qPCR assays simultaneously in a high-density array format. We measured the transcription from two different adult human tissues to assess measurement reproducibility across replicates, measurement accuracy, precision, specificity, and sensitivity; determined the false positive rate (FPR) and false negative rate (FNR) of the expressed transcript copies; and determined differences in kinase gene expression reflecting tissue and dosage differences. Using our methodology, we confirm the potential of this technology in advancing pharmaceutical research and development.


Assuntos
Nanotecnologia/métodos , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Reações Falso-Negativas , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Proteínas de Fluorescência Verde/metabolismo , Humanos , Fígado/metabolismo , Miocárdio/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Análise de Componente Principal
13.
Biotechniques ; 46(3 Suppl): ix-xiii, 2009 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-19317669

RESUMO

Discovery, evaluation, and understanding the biological relevance of single nucleotide polymorphisms (SNPs) and their associated phenotypes is relevant to many applications, including human disease diagnostics, pathogen detection, and identification of genetic traits impacting agricultural practices, both in terms of food quality and production efficiency. Validation of putative SNP associations in large-scale cohorts is currently impeded by the technical challenges and high cost inherent in analyzing large numbers of samples using available SNP genotyping platforms. We describe in this report the implementation of the 5'-exonuclease, biallelic PCR assay for SNP genotyping (TaqMan) in a nanofluidic version of a high-density microplate. System performance was assessed using a panel of 32 TaqMan SNP genotyping assays targeted to human polymorphisms. This functional test of the nanoliter fluidic SNP genotyping platform delivered genotyping call rates and accuracies comparable to the same larger volume reactions in microplate systems.


Assuntos
Nanotecnologia , Reação em Cadeia da Polimerase , Polimorfismo de Nucleotídeo Único , Genótipo , Humanos , Nanotecnologia/instrumentação , Nanotecnologia/métodos , Reprodutibilidade dos Testes
14.
Methods Mol Biol ; 496: 161-74, 2009.
Artigo em Inglês | MEDLINE | ID: mdl-18839111

RESUMO

The increasing emphasis in life science research on utilization of genetic and genomic information underlies the need for high-throughput technologies capable of analyzing the expression of multiple genes or the presence of informative single nucleotide polymorphisms (SNPs) in large-scale, population-based applications. Human disease research, disease diagnosis, personalized therapeutics, environmental monitoring, blood testing, and identification of genetic traits impacting agricultural practices, both in terms of food quality and production efficiency, are a few areas where such systems are in demand. This has stimulated the need for PCR technologies that preserves the intrinsic analytical benefits of PCR yet enables higher throughputs without increasing the time to answer, labor and reagent expenses and workflow complexity. An example of such a system based on a high-density array of nanoliter PCR assays is described here. Functionally equivalent to a microtiter plate, the nanoplate system makes possible up to 3,072 simultaneous end-point or real-time PCR measurements in a device, the size of a standard microscope slide. Methods for SNP genotyping with end-point TaqMan PCR assays and quantitative measurement of gene expression with SYBR Green I real-time PCR are outlined and illustrative data showing system performance is provided.


Assuntos
DNA/genética , Perfilação da Expressão Gênica/métodos , Reação em Cadeia da Polimerase/métodos , Polimorfismo de Nucleotídeo Único , RNA/genética , Animais , Humanos , Sensibilidade e Especificidade
15.
Nucleic Acids Res ; 34(18): e123, 2006.
Artigo em Inglês | MEDLINE | ID: mdl-17000636

RESUMO

Understanding biological complexity arising from patterns of gene expression requires accurate and precise measurement of RNA levels across large numbers of genes simultaneously. Real time PCR (RT-PCR) in a microtiter plate is the preferred method for quantitative transcriptional analysis but scaling RT-PCR to higher throughputs in this fluidic format is intrinsically limited by cost and logistic considerations. Hybridization microarrays measure the transcription of many thousands of genes simultaneously yet are limited by low sensitivity, dynamic range, accuracy and sample throughput. The hybrid approach described here combines the superior accuracy, precision and dynamic range of RT-PCR with the parallelism of a microarray in an array of 3072 real time, 33 nl polymerase chain reactions (RT-PCRs) the size of a microscope slide. RT-PCR is demonstrated with an accuracy and precision equivalent to the same assay in a 384-well microplate but in a 64-fold smaller reaction volume, a 24-fold higher analytical throughput and a workflow compatible with standard microplate protocols.


Assuntos
Perfilação da Expressão Gênica/métodos , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Reação em Cadeia da Polimerase/métodos , Células Cultivadas , Endotélio Vascular/citologia , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/metabolismo , Humanos , Fígado/enzimologia , Miocárdio/enzimologia , Fosfotransferases/biossíntese , Fosfotransferases/genética , RNA Mensageiro/metabolismo , Reprodutibilidade dos Testes , Fator de Necrose Tumoral alfa/farmacologia
16.
Drug Discov Today Technol ; 2(3): 247-53, 2005.
Artigo em Inglês | MEDLINE | ID: mdl-24981943

RESUMO

The recent completion of the human genome sequence has increased the need for high throughput quantitative transcription analysis. Quantitative PCR is an alternative to microarrays for accurate and precise expression analysis with single transcript copy sensitivity. A review of current research in miniaturized, high throughput qPCR suggests this technique will soon be a viable option to hybridization microarrays for large-scale genetic analyses.:

17.
Assay Drug Dev Technol ; 2(4): 373-81, 2004 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-15357918

RESUMO

Mass spectrometry-based screening can be applied to a wide range of targets, including those intractable targets that use substrates such as lipids, fatty acids, phospholipids, steroids, prostaglandins, and other compounds not generally amenable to conventional screening techniques. The major limitation to this approach is throughput, making HTS via mass spectrometry impractical. We present a mass spectrometry-based technique and hardware for lead discovery applications. Mass spectrometry enables the design of label-free assays using biologically native substrates for a wide range of enzymatic targets. This system can be used for the direct quantification of analytes in complex reaction mixtures with typical throughputs of 4-5 s per sample. A mass spectrometry-based assay was developed to identify inhibitors of acetylcholinesterase, an enzyme with clinical importance in Alzheimer's disease. The system was used to screen a small chemical library. Several potent inhibitors were identified, and the IC(50) values of the inhibitors were determined.


Assuntos
Acetilcolinesterase/análise , Acetilcolinesterase/metabolismo , Técnicas Analíticas Microfluídicas/métodos , Inibidores da Colinesterase/química , Inibidores da Colinesterase/farmacologia , Humanos , Concentração Inibidora 50 , Espectrometria de Massas/métodos , Técnicas Analíticas Microfluídicas/instrumentação
18.
J Pharm Sci ; 92(1): 149-60, 2003 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-12486691

RESUMO

Knowledge and control of the polymorphic phase of chemical compounds are important aspects of drug development in the pharmaceutical industry. We report herein in situ and real-time Raman spectroscopic polymorphic analysis of optically trapped microcrystals in a microliter volume format. The system studied in particular was the recrystallization of carbamazepine (CBZ) in methanol. Raman spectrometry enabled noninvasive measurement of the amount of dissolved CBZ in a sample as well as polymorphic characterization, whereas exclusive recrystallization of either CBZ form I or CBZ form III from saturated solutions was achieved by specific selection of sample cell cooling profiles. Additionally, using a microcell versus a macroscopic volume gives the advantage of reaching equilibrium much faster while using little compound quantity. We demonstrate that laser Raman spectral polymorphic analysis in a microliter cell is a potentially viable screening platform for polymorphic analysis and could lead to a new high throughput method for polymorph screening.


Assuntos
Soluções Farmacêuticas/análise , Tecnologia Farmacêutica/métodos , Cristalização , Lasers , Soluções Farmacêuticas/química , Análise Espectral Raman/instrumentação , Análise Espectral Raman/métodos , Tecnologia Farmacêutica/instrumentação
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