Bacterial Single Cell Whole Transcriptome Amplification in Microfluidic Platform Shows Putative Gene Expression Heterogeneity.
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ABSTRACT:
SUBMITTER: Liu Y
PROVIDER: S-EPMC8422856 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Single-cell whole-transcriptome analysis is a powerful tool for quantifying gene expression heterogeneity in populations of cells. Many techniques have, thus, been recently developed to perform transcriptome sequencing (RNA-Seq) on individual cells. To probe subtle biological variation between samples with limiting amounts of RNA, more precise and sensitive methods are still required. We adapted a previously developed strategy for single-cell RNA-Seq that has shown promise for superior sensitivity and implemented the chemistry in a microfluidic platform for single-cell whole-transcriptome analysis. In this approach, single cells are captured and lysed in a microfluidic device, where mRNAs with poly(A) tails are reverse-transcribed into cDNA. Double-stranded cDNA is then collected and sequenced using a next generation sequencing platform. We prepared 94 libraries consisting of single mouse embryonic cells and technical replicates of extracted RNA and thoroughly characterized the performance of this technology. Microfluidic implementation increased mRNA detection sensitivity as well as improved measurement precision compared with tube-based protocols. With 0.2 M reads per cell, we were able to reconstruct a majority of the bulk transcriptome with 10 single cells. We also quantified variation between and within different types of mouse embryonic cells and found that enhanced measurement precision, detection sensitivity, and experimental throughput aided the distinction between biological variability and technical noise. With this work, we validated the advantages of an early approach to single-cell RNA-Seq and showed that the benefits of combining microfluidic technology with high-throughput sequencing will be valuable for large-scale efforts in single-cell transcriptome analysis.
| S-EPMC4024894 | biostudies-literature
Project description:Single-cell whole-transcriptome analysis is a powerful tool for quantifying gene expression heterogeneity in populations of cells. Many techniques have, thus, been recently developed to perform transcriptome sequencing (RNA-Seq) on individual cells. To probe subtle biological variation between samples with limiting amounts of RNA, more precise and sensitive methods are still required. We adapted a previously developed strategy for single-cell RNA-Seq that has shown promise for superior sensitivity and implemented the chemistry in a microfluidic platform for single-cell whole transcriptome analysis. In this approach, single cells are captured and lysed in a microfluidic device, where mRNAs with poly(A) tails are reverse-transcribed into cDNA. Double-stranded cDNA is then collected and sequenced using a next-generation sequencing platform. We prepared 94 libraries consisting of single mouse embryonic cells and technical replicates of extracted RNA and thoroughly characterized the performance of this technology. Microfluidic implementation increased mRNA detection sensitivity as well as improved measurement precision compared with tube-based protocols. With 0.2M reads per cell, we were able to reconstruct a majority of the bulk transcriptome with 10 single cells. We also quantified variation between and within different types of mouse embryonic cells and found that enhanced measurement precision, detection sensitivity, and experimental throughput aided the distinction between biological variability and technical noise. With this work, we validated the advantages of an early approach to single-cell RNA-Seq and showed that the benefits of combining microfluidic technology with high-throughput sequencing will be valuable for large-scale efforts in single-cell transcriptome analysis. We investigated gene expression in mouse embryonic cells using microfluidic-facilitated RNA-Seq to analyze 56 single mouse ES cell (mESC) transcriptomes and 6 single mouse embryonic fibroblast (MEF) transcriptomes. To quantitatively evaluate the sensitivity and precision of our technique, we also sequenced 23 libraries from extracted mESC RNA, representing three sets of technical replicates with varying starting amounts of material.
2014-05-01 | E-GEOD-47835 | biostudies-arrayexpress
Project description:Single-cell sequencing is a powerful technology that provides the capability of analyzing a single cell within a population. This technology is mostly coupled with microfluidic systems for controlled cell manipulation and precise fluid handling to shed light on the genomes of a wide range of cells. So far, single-cell sequencing has been focused mostly on human cells due to the ease of lysing the cells for genome amplification. The major challenges that bacterial species pose to genome amplification from single cells include the rigid bacterial cell walls and the need for an effective lysis protocol compatible with microfluidic platforms. In this work, we present a lysis protocol that can be used to extract genomic DNA from both gram-positive and gram-negative species without interfering with the amplification chemistry. Corynebacterium glutamicum was chosen as a typical gram-positive model and Nostoc sp. as a gram-negative model due to major challenges reported in previous studies. Our protocol is based on thermal and chemical lysis. We consider 80% of single-cell replicates that lead to >5 ng DNA after amplification as successful attempts. The protocol was directly applied to Gloeocapsa sp. and the single cells of the eukaryotic Sphaerocystis sp. and achieved a 100% success rate.
| S-EPMC6187716 | biostudies-literature
Project description:Single Bacterial Cell Whole Genome Amplification
| PRJEB2063 | ENA
Project description:Single-cell whole-transcriptome analysis is a powerful tool for quantifying gene expression heterogeneity in populations of cells. Many techniques have, thus, been recently developed to perform transcriptome sequencing (RNA-Seq) on individual cells. To probe subtle biological variation between samples with limiting amounts of RNA, more precise and sensitive methods are still required. We adapted a previously developed strategy for single-cell RNA-Seq that has shown promise for superior sensitivity and implemented the chemistry in a microfluidic platform for single-cell whole transcriptome analysis. In this approach, single cells are captured and lysed in a microfluidic device, where mRNAs with poly(A) tails are reverse-transcribed into cDNA. Double-stranded cDNA is then collected and sequenced using a next-generation sequencing platform. We prepared 94 libraries consisting of single mouse embryonic cells and technical replicates of extracted RNA and thoroughly characterized the performance of this technology. Microfluidic implementation increased mRNA detection sensitivity as well as improved measurement precision compared with tube-based protocols. With 0.2M reads per cell, we were able to reconstruct a majority of the bulk transcriptome with 10 single cells. We also quantified variation between and within different types of mouse embryonic cells and found that enhanced measurement precision, detection sensitivity, and experimental throughput aided the distinction between biological variability and technical noise. With this work, we validated the advantages of an early approach to single-cell RNA-Seq and showed that the benefits of combining microfluidic technology with high-throughput sequencing will be valuable for large-scale efforts in single-cell transcriptome analysis.
2014-05-01 | GSE47835 | GEO
Project description:BackgroundSingle-cell genome sequencing has the potential to allow the in-depth exploration of the vast genetic diversity found in uncultured microbes. We used the marine cyanobacterium Prochlorococcus as a model system for addressing important challenges facing high-throughput whole genome amplification (WGA) and complete genome sequencing of individual cells.Methodology/principal findingsWe describe a pipeline that enables single-cell WGA on hundreds of cells at a time while virtually eliminating non-target DNA from the reactions. We further developed a post-amplification normalization procedure that mitigates extreme variations in sequencing coverage associated with multiple displacement amplification (MDA), and demonstrated that the procedure increased sequencing efficiency and facilitated genome assembly. We report genome recovery as high as 99.6% with reference-guided assembly, and 95% with de novo assembly starting from a single cell. We also analyzed the impact of chimera formation during MDA on de novo assembly, and discuss strategies to minimize the presence of incorrectly joined regions in contigs.Conclusions/significanceThe methods describe in this paper will be useful for sequencing genomes of individual cells from a variety of samples.
| S-EPMC2731171 | biostudies-literature
Project description:Cytotoxicity is a vital effector mechanism used by immune cells to combat pathogens and cancer cells. While conventional cytotoxicity assays rely on averaged end-point measures, crucial insights on the dynamics and heterogeneity of effector and target cell interactions cannot be extracted, emphasizing the need for dynamic single-cell analysis. Here, we present a fully automated droplet-based microfluidic platform that allowed the real-time monitoring of effector-target cell interactions and killing, allowing the screening of over 60,000 droplets identifying 2000 individual cellular interactions monitored over 10 h. During the course of incubation, we observed that the dynamics of cytotoxicity within the Natural Killer (NK) cell population varies significantly over the time. Around 20% of the total NK cells in droplets showed positive cytotoxicity against paired K562 cells, most of which was exhibited within first 4 h of cellular interaction. Using our single cell analysis platform, we demonstrated that the population of NK cells is composed of individual cells with different strength in their effector functions, a behavior masked in conventional studies. Moreover, the versatility of our platform will allow the dynamic and resolved study of interactions between immune cell types and the finding and characterization of functional sub-populations, opening novel ways towards both fundamental and translational research.
| S-EPMC8385055 | biostudies-literature
Project description:Neutrophils play an essential role in protection against infections and their numbers in the blood are frequently measured in the clinic. Higher neutrophil counts in the blood are usually an indicator of ongoing infections, while low neutrophil counts are a warning sign for higher risks for infections. To accomplish their functions, neutrophils also have to be able to move effectively from the blood where they spend most of their life, into tissues, where infections occur. Consequently, any defects in the ability of neutrophils to migrate can increase the risks for infections, even when neutrophils are present in appropriate numbers in the blood. However, measuring neutrophil migration ability in the clinic is a challenging task, which is time consuming, requires large volume of blood, and expert knowledge. To address these limitations, we designed a robust microfluidic assays for neutrophil migration, which requires a single droplet of unprocessed blood, circumvents the need for neutrophil separation, and is easy to quantify on a simple microscope. In this assay, neutrophils migrate directly from the blood droplet, through small channels, towards the source of chemoattractant. To prevent the granular flow of red blood cells through the same channels, we implemented mechanical filters with right angle turns that selectively block the advance of red blood cells. We validated the assay by comparing neutrophil migration from blood droplets collected from finger prick and venous blood. We also compared these whole blood (WB) sources with neutrophil migration from samples of purified neutrophils and found consistent speed and directionality between the three sources. This microfluidic platform will enable the study of human neutrophil migration in the clinic and the research setting to help advance our understanding of neutrophil functions in health and disease.
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Project description:Understanding transcriptome complexity is crucial for understanding human biology and disease. Technologies such as Synthetic long-read RNA sequencing (SLR-RNA-seq) delivered 5 million isoforms and allowed assessing splicing coordination. Pacific Biosciences and Oxford Nanopore increase throughput also but require high input amounts or amplification. Our new droplet-based method, sparse isoform sequencing (spISO-seq), sequences 100k-200k partitions of 10-200 molecules at a time, enabling analysis of 10-100 million RNA molecules. SpISO-seq requires less than 1 ng of input cDNA, limiting or removing the need for prior amplification with its associated biases. Adjusting the number of reads devoted to each molecule reduces sequencing lanes and cost, with little loss in detection power. The increased number of molecules expands our understanding of isoform complexity. In addition to confirming our previously published cases of splicing coordination (e.g., BIN1), the greater depth reveals many new cases, such as MAPT Coordination of internal exons is found to be extensive among protein coding genes: 23.5%-59.3% (95% confidence interval) of highly expressed genes with distant alternative exons exhibit coordination, showcasing the need for long-read transcriptomics. However, coordination is less frequent for noncoding sequences, suggesting a larger role of splicing coordination in shaping proteins. Groups of genes with coordination are involved in protein-protein interactions with each other, raising the possibility that coordination facilitates complex formation and/or function. We also find new splicing coordination types, involving initial and terminal exons. Our results provide a more comprehensive understanding of the human transcriptome and a general, cost-effective method to analyze it.
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Project description:Antigen-specific T cells play an essential role in immunoregulation and many diseases such as cancer. Characterizing the T cell receptor (TCR) sequences that encode T cell specificity is critical for elucidating the antigenic determinants of immunological diseases and designing therapeutic remedies. However, methods of obtaining single-cell TCR sequencing data are labor and cost intensive, typically requiring both cell sorting and full-length single-cell RNA-sequencing (scRNA-seq). New high-throughput 3' cell-barcoding scRNA-seq methods can simplify and scale this process; however, they do not routinely capture TCR sequences during library preparation and sequencing. While 5' cell-barcoding scRNA-seq methods can be used to examine TCR repertoire at single-cell resolution, doing so requires specialized reagents which cannot be applied to samples previously processed using 3' cell-barcoding methods.Here, we outline a method for sequencing TCRα and TCRβ transcripts from samples already processed using 3' cell-barcoding scRNA-seq platforms, ensuring TCR recovery at a single-cell resolution. In short, a fraction of the 3' barcoded whole transcriptome amplification (WTA) product typically used to generate a massively parallel 3' scRNA-seq library is enriched for TCR transcripts using biotinylated probes and further amplified using the same universal primer sequence from WTA. Primer extension using TCR V-region primers and targeted PCR amplification using a second universal primer result in a 3' barcoded single-cell CDR3-enriched library that can be sequenced with custom sequencing primers. Coupled with 3' scRNA-seq of the same WTA, this method enables simultaneous analysis of single-cell transcriptomes and TCR sequences which can help interpret inherent heterogeneity among antigen-specific T cells and salient disease biology. The method presented here can also be adapted readily to enrich and sequence other transcripts of interest from both 3' and 5' barcoded scRNA-seq WTA libraries.
| S-EPMC10283041 | biostudies-literature