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Spontaneous mutations introduce uncertainty into coronavirus disease 2019 (COVID-19) control procedures and vaccine development. Here, we perform a spatiotemporal analysis on intra-host single-nucleotide variants (iSNVs) in 402 clinical samples from 170 affected individuals, which reveals an increase in genetic diversity over time after symptom onset in individuals. Nonsynonymous mutations are overrepresented in the pool of iSNVs but underrepresented at the single-nucleotide polymorphism (SNP) level, suggesting a two-step fitness selection process: a large number of nonsynonymous substitutions are generated in the host (positive selection), and these substitutions tend to be unfixed as SNPs in the population (negative selection). Dynamic iSNV changes in subpopulations with different gender, age, illness severity, and viral shedding time displayed a varied fitness selection process among populations. Our study highlights that iSNVs provide a mutational pool shaping the rapid global evolution of the virus.
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COVID-19/virología , Interacciones Huésped-Patógeno/genética , SARS-CoV-2/genética , Adolescente , Adulto , Anciano , Niño , Preescolar , Femenino , Genoma Viral/genética , Humanos , Lactante , Recién Nacido , Masculino , Persona de Mediana Edad , Mutación/genética , Filogenia , Polimorfismo de Nucleótido Simple/genética , Glicoproteína de la Espiga del Coronavirus/genética , Desarrollo de Vacunas/métodos , Adulto JovenRESUMEN
After a short recovery period, COVID-19 reinfections could occur in convalescent patients, even those with measurable levels of neutralizing antibodies. Effective vaccinations and protective public health measures are recommended for the convalescent COVID-19 patients.
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A decade since its invention, single-cell RNA sequencing (scRNA-seq) has become a mainstay technology for profiling transcriptional heterogeneity in individual cells. Yet, most existing scRNA-seq methods capture only polyadenylated mRNA to avoid the cost of sequencing non-messenger transcripts, such as ribosomal RNA (rRNA), that are usually not of-interest. Hence, there are not very many protocols that enable single-cell analysis of total RNA. We adapted a method called DASH (Depletion of Abundant Sequences by Hybridisation) to make it suitable for depleting rRNA sequences from single-cell total RNA-seq libraries. Our analyses show that our single-cell DASH (scDASH) method can effectively deplete rRNAs from sequencing libraries with minimal off-target non-specificity. Importantly, as a result of depleting the rRNA, the rest of the transcriptome is significantly enriched for detection.
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Emulsion polymerase chain reaction (ePCR) enables parallel amplification of millions of different DNA molecules while avoiding bias and chimeric byproducts, essential criteria for applications including next generation sequencing, aptamer selection, and protein-DNA interaction studies. Despite these advantages, ePCR remains underused due to the lack of optimal starting conditions, straightforward methods to evaluate success, and guidelines for tuning the reaction. This knowledge has been elusive for bulk emulsion generation methods, such as stirring and vortexing, the only methods that can emulsify libraries of ≥108 sequences within minutes, because these emulsions have not been characterized in ways that preserve the heterogeneity that defines successful ePCR. Our study quantifies the outcome of ePCR from conditions specified in the literature using single particle analysis, which preserves this heterogeneity. We combine ePCR with magnetic microbeads and quantify the amplification yield via qPCR and the proportion of clonal and saturated beads via flow cytometry. Our single particle level analysis of thousands of beads resolves two key criteria that define the success of ePCR: 1) whether the target fraction of 20% clonal beads predicted by the Poisson distribution is achieved, and 2) whether those beads are partially or maximally covered by amplified DNA. We found that among the two concentrations of polymerase tested, only the higher one, which is 20-fold more than the concentration recommended for conventional PCR, could yield sufficient PCR products. Dramatic increases in the concentrations of reverse primer and nucleotides recommended in literature gave no measurable change in outcome. We thus provide evidence-based starting conditions for effective and economical ePCR for real DNA libraries and a straightforward workflow for evaluating the success of tuning ePCR prior to downstream applications.
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Imagen Individual de Molécula , Cartilla de ADN , Emulsiones , Biblioteca de Genes , Reacción en Cadena de la PolimerasaRESUMEN
Analyzing the genome of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) from clinical samples is crucial for understanding viral spread and evolution as well as for vaccine development. Existing RNA sequencing methods are demanding on user technique and time and, thus, not ideal for time-sensitive clinical samples; these methods are also not optimized for high performance on viral genomes. We developed a facile, practical, and robust approach for metagenomic and deep viral sequencing from clinical samples. We demonstrate the utility of our approach on pharyngeal, sputum, and stool samples collected from coronavirus disease 2019 (COVID-19) patients, successfully obtaining whole metatranscriptomes and complete high-depth, high-coverage SARS-CoV-2 genomes with high yield and robustness. With a shortened hands-on time from sample to virus-enriched sequencing-ready library, this rapid, versatile, and clinic-friendly approach will facilitate molecular epidemiology studies during current and future outbreaks.
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COVID-19/genética , Genoma Viral , Secuenciación de Nucleótidos de Alto Rendimiento , ARN Viral/genética , SARS-CoV-2/genética , Secuenciación Completa del Genoma , Animales , Humanos , Ratones , Células 3T3 NIH , ARN Viral/metabolismo , SARS-CoV-2/metabolismoRESUMEN
The spread of SARS-CoV-2 in Beijing before May, 2020 resulted from transmission following both domestic and global importation of cases. Here we present genomic surveillance data on 102 imported cases, which account for 17.2% of the total cases in Beijing. Our data suggest that all of the cases in Beijing can be broadly classified into one of three groups: Wuhan exposure, local transmission and overseas imports. We classify all sequenced genomes into seven clusters based on representative high-frequency single nucleotide polymorphisms (SNPs). Genomic comparisons reveal higher genomic diversity in the imported group compared to both the Wuhan exposure and local transmission groups, indicating continuous genomic evolution during global transmission. The imported group show region-specific SNPs, while the intra-host single nucleotide variations present as random features, and show no significant differences among groups. Epidemiological data suggest that detection of cases at immigration with mandatory quarantine may be an effective way to prevent recurring outbreaks triggered by imported cases. Notably, we also identify a set of novel indels. Our data imply that SARS-CoV-2 genomes may have high mutational tolerance.
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Betacoronavirus/crecimiento & desarrollo , Infecciones por Coronavirus/virología , Neumonía Viral/virología , Adulto , Beijing/epidemiología , COVID-19 , Infecciones por Coronavirus/epidemiología , Femenino , Genoma Viral , Genómica , Genotipo , Humanos , Masculino , Persona de Mediana Edad , Mutación , Pandemias , Filogenia , Neumonía Viral/epidemiología , Polimorfismo de Nucleótido Simple , SARS-CoV-2 , Viaje , Adulto JovenRESUMEN
Despite being a relatively recent technological development, single-cell transcriptional analysis through high-throughput sequencing has already been used in hundreds of fruitful studies to make exciting new biological discoveries that would otherwise be challenging or even impossible. Consequently, this has fueled a virtuous cycle of even greater interest in the field and compelled development of further improved technical methodologies and approaches. Thanks to the combined efforts of the research community, including the fields of biochemistry and molecular biology, technology and instrumentation, data science, computational biology, and bioinformatics, the single-cell RNA-sequencing field is advancing at a pace that is both astounding and unprecedented. In this review, we provide a broad introduction to this revolutionary technology by presenting the state-of-the-art in sample preparation methodologies, technology platforms, and computational analysis methods, while highlighting the key considerations for designing, executing, and interpreting a study using single-cell RNA sequencing.
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ADN/análisis , Análisis de la Célula Individual/métodos , Animales , ADN/química , ADN/metabolismo , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Microfluídica , Reacción en Cadena de la Polimerasa , Empalme del ARN , Análisis de Secuencia de ARNRESUMEN
Protein-DNA interactions are responsible for numerous critical cellular events: For example, gene expression and silencing are mediated by transcription factor protein binding and histone protein modifications, and DNA replication and repair rely on site-specific protein binding. Chromatin immunoprecipitation (ChIP) is the only molecular assay that directly determines, in a living cell, the binding association between a protein of interest and specific genomic loci. It is an indispensible tool in the biologist's toolbox, but the many limitations of this technique prevent broad adoption of ChIP in biological studies. The typical ChIP assay can take up to 1 wk to complete, and the process is technically tricky, yet tedious. The ChIP assay yields are also low, thus requiring on the order of millions to billions of cells as starting material, which makes the assay unfeasible for studies using rare or precious samples. For example, fluorescence-activated cell sorting (FACS) of cancer stem cells (CSCs) obtained from primary tumors, rarely yields more than ~100,000 CSCs per tumor. This protocol describes a microfluidics-based strategy for performing ChIP, which uses automation and scalability to reduce both total and hands-on assay time, and improve throughput. It allows whole fixed cells as input, and enables automated ChIP from as few as 2000 cells.
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Inmunoprecipitación de Cromatina/métodos , Microfluídica/métodosRESUMEN
Individual mammalian cells exhibit large variability in cellular volume, even with the same absolute DNA content, and so must compensate for differences in DNA concentration in order to maintain constant concentration of gene expression products. Using single-molecule counting and computational image analysis, we show that transcript abundance correlates with cellular volume at the single-cell level due to increased global transcription in larger cells. Cell fusion experiments establish that increased cellular content itself can directly increase transcription. Quantitative analysis shows that this mechanism measures the ratio of cellular volume to DNA content, most likely through sequestration of a transcriptional factor to DNA. Analysis of transcriptional bursts reveals a separate mechanism for gene dosage compensation after DNA replication that enables proper transcriptional output during early and late S phase. Our results provide a framework for quantitatively understanding the relationships among DNA content, cell size, and gene expression variability in single cells.
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Dosificación de Gen , Hibridación Fluorescente in Situ/métodos , Análisis de Secuencia de ARN/métodos , Análisis de la Célula Individual/métodos , Transcripción Genética , Animales , Caenorhabditis elegans/genética , Células Cultivadas , Fibroblastos/citología , Prepucio/citología , Expresión Génica , Humanos , Masculino , Datos de Secuencia Molecular , Fase SRESUMEN
The mammalian lung is a highly branched network in which the distal regions of the bronchial tree transform during development into a densely packed honeycomb of alveolar air sacs that mediate gas exchange. Although this transformation has been studied by marker expression analysis and fate-mapping, the mechanisms that control the progression of lung progenitors along distinct lineages into mature alveolar cell types are still incompletely known, in part because of the limited number of lineage markers and the effects of ensemble averaging in conventional transcriptome analysis experiments on cell populations. Here we show that single-cell transcriptome analysis circumvents these problems and enables direct measurement of the various cell types and hierarchies in the developing lung. We used microfluidic single-cell RNA sequencing (RNA-seq) on 198 individual cells at four different stages encompassing alveolar differentiation to measure the transcriptional states which define the developmental and cellular hierarchy of the distal mouse lung epithelium. We empirically classified cells into distinct groups by using an unbiased genome-wide approach that did not require a priori knowledge of the underlying cell types or the previous purification of cell populations. The results confirmed the basic outlines of the classical model of epithelial cell-type diversity in the distal lung and led to the discovery of many previously unknown cell-type markers, including transcriptional regulators that discriminate between the different populations. We reconstructed the molecular steps during maturation of bipotential progenitors along both alveolar lineages and elucidated the full life cycle of the alveolar type 2 cell lineage. This single-cell genomics approach is applicable to any developing or mature tissue to robustly delineate molecularly distinct cell types, define progenitors and lineage hierarchies, and identify lineage-specific regulatory factors.
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Linaje de la Célula/genética , Células Epiteliales/citología , Células Epiteliales/metabolismo , Pulmón/citología , Análisis de Secuencia de ARN/métodos , Análisis de la Célula Individual/métodos , Animales , Bronquios/citología , Diferenciación Celular/genética , Células Epiteliales/clasificación , Femenino , Marcadores Genéticos , Genoma/genética , Genómica , Pulmón/embriología , Ratones , Ratones Endogámicos C57BL , Alveolos Pulmonares/citología , Intercambio Gaseoso Pulmonar , Células Madre/citología , Transcriptoma/genéticaRESUMEN
Interest in single-cell whole-transcriptome analysis is growing rapidly, especially for profiling rare or heterogeneous populations of cells. We compared commercially available single-cell RNA amplification methods with both microliter and nanoliter volumes, using sequence from bulk total RNA and multiplexed quantitative PCR as benchmarks to systematically evaluate the sensitivity and accuracy of various single-cell RNA-seq approaches. We show that single-cell RNA-seq can be used to perform accurate quantitative transcriptome measurement in individual cells with a relatively small number of sequencing reads and that sequencing large numbers of single cells can recapitulate bulk transcriptome complexity.
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Análisis de Secuencia de ARN/métodos , Análisis de la Célula Individual/métodos , Interpretación Estadística de Datos , Procesamiento Automatizado de Datos , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Células HCT116 , Humanos , Microfluídica , Técnicas de Amplificación de Ácido Nucleico , Reacción en Cadena de la Polimerasa/métodos , Reproducibilidad de los Resultados , Análisis de Secuencia de ADN , TranscriptomaRESUMEN
Chromatin immunoprecipitation (ChIP) is an assay for interrogating protein-DNA interactions that is increasingly being used for drug target discovery and screening applications. Currently the complexity of the protocol and the amount of hands-on time required for this assay limits its use to low throughput applications; furthermore, variability in antibody quality poses an additional obstacle in scaling up ChIP for large scale screening purposes. To address these challenges, we report HTChIP, an automated microfluidic-based platform for performing high-throughput ChIP screening measurements of 16 different targets simultaneously, with potential for further scale-up. From chromatin to analyzable PCR results only takes one day using HTChIP, as compared to several days up to one week for conventional protocols. HTChIP can also be used to test multiple antibodies and select the best performer for downstream ChIP applications, saving time and reagent costs of unsuccessful ChIP assays as a result of poor antibody quality. We performed a series of characterization assays to demonstrate that HTChIP can rapidly and accurately evaluate the epigenetic states of a cell, and that it is sensitive enough to detect the changes in the epigenetic state induced by a cytokine stimulant over a fine temporal resolution. With these results, we believe that HTChIP can introduce large improvements in routine ChIP, antibody screening, and drug screening efficiency, and further facilitate the use of ChIP as a valuable tool for research and discovery.
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Anticuerpos/inmunología , Inmunoprecipitación de Cromatina , Automatización , Proliferación Celular/efectos de los fármacos , Evaluación Preclínica de Medicamentos , Células HeLa , Ensayos Analíticos de Alto Rendimiento , Histonas/metabolismo , Humanos , Técnicas Analíticas Microfluídicas , Factor de Necrosis Tumoral alfa/metabolismo , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
The sirtuin Sirt6 is a NAD-dependent histone deacetylase that is implicated in gene regulation and lifespan control. Sirt6 can interact with the stress-responsive transcription factor NF-κB and regulate some NF-κB target genes, but the full scope of Sirt6 target genes as well as dynamics of Sirt6 occupancy on chromatin are not known. Here we map Sirt6 occupancy on mouse promoters genome-wide and show that Sirt6 occupancy is highly dynamic in response to TNF-α. More than half of Sirt6 target genes are only revealed upon stress-signaling. The majority of genes bound by NF-κB subunit RelA recruit Sirt6, and dynamic Sirt6 relocalization is largely driven in a RelA-dependent manner. Integrative analysis with global gene expression patterns in wild-type, Sirt6-/-, and double Sirt6-/- RelA-/- cells reveals the epistatic relationships between Sirt6 and RelA in shaping diverse temporal patterns of gene expression. Genes under the direct joint control of Sirt6 and RelA include several with prominent roles in cell senescence and organismal aging. These data suggest dynamic chromatin relocalization of Sirt6 as a key output of NF-κB signaling in stress response and aging.
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Envejecimiento/genética , Cromatina/metabolismo , Redes Reguladoras de Genes/genética , Sirtuinas/metabolismo , Estrés Fisiológico/genética , Animales , Línea Celular , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Espacio Intracelular/metabolismo , Ratones , Ratones Noqueados , Modelos Genéticos , Transporte de Proteínas/genética , Reproducibilidad de los Resultados , Factor de Transcripción ReIA/metabolismoRESUMEN
Chromatin immunoprecipitation (ChIP) is a powerful assay used to probe DNA-protein interactions. Traditional methods of implementing this assay are lengthy, cumbersome and require a large number of cells, making it difficult to study rare cell types such as certain cancer and stem cells. We have designed a microfluidic device to perform sensitive ChIP analysis on low cell numbers in a rapid, automated fashion while preserving the specificity of the assay. Comparing ChIP results for two modified histone protein targets, we showed our automated microfluidic ChIP (AutoChIP) from 2,000 cells to be comparable to that of conventional ChIP methods using 50,000-500,000 cells. This technology may provide a solution to the need for a high sensitivity, rapid, and automated ChIP assay, and in doing so facilitate the use of ChIP for many interesting and valuable applications.
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Inmunoprecipitación de Cromatina , Técnicas Analíticas Microfluídicas , Animales , Automatización , Recuento de Células , Línea Celular Tumoral , Inmunoprecipitación de Cromatina/instrumentación , Inmunoprecipitación de Cromatina/métodos , Diseño de Equipo , Regulación Neoplásica de la Expresión Génica , Histonas/química , Ratones , Técnicas Analíticas Microfluídicas/instrumentación , Técnicas Analíticas Microfluídicas/métodosRESUMEN
A novel open-access microfluidic patch-clamp array chip with lateral cell trapping sites raised above the bottom plane of the chip was developed by combining both a microscale soft-lithography and a macroscale polymer fabrication method. This paper demonstrates the capability of using such an open-access fluidic system for patch-clamp measurements. The surface of the open-access patch-clamp sites prepared by the macroscale hole patterning method of soft-state elastic polydimethylsiloxane (PDMS) is examined; the seal resistances are characterized and correlated with the aperture dimensions. Whole cell patch-clamp measurements are carried out with CHO cells expressing Kv2.1 ion channels. Kv2.1 ion channel blocker (TEA) dosage response is characterized and the binding activity is examined. The results demonstrate that the system is capable of performing whole cell measurements and drug profiling in a more efficient manner than the traditional patch-clamp set-up.