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Whole Genome Amplification and De novo Assembly of Single Bacterial Cells  [PDF]
Sébastien Rodrigue, Rex R. Malmstrom, Aaron M. Berlin, Bruce W. Birren, Matthew R. Henn, Sallie W. Chisholm
PLOS ONE , 2009, DOI: 10.1371/journal.pone.0006864
Abstract: Background Single-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 Findings We 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/Significance The methods describe in this paper will be useful for sequencing genomes of individual cells from a variety of samples.
Evolution of oxygen utilization in multicellular organisms and implications for cell signalling in tissue engineering
Katerina Stamati,Vivek Mudera,Umber Cheema
Journal of Tissue Engineering , 2011, DOI: 10.1177/2041731411432365
Abstract: Oxygen is one of the critically defining elements resulting in the existence of eukaryotic life on this planet. The rise and fall of this element can be tracked through time and corresponds with the evolution of diverse life forms, development of efficient energy production (oxidative phosphorylation) in single cell organisms, the evolution of multicellular organisms and the regulation of complex cell phenotypes. By understanding these events, we can plot the effect of oxygen on evolution and its direct influence on different forms of life today, from the whole organism to specific cells within multicellular organisms. In the emerging field of tissue engineering, understanding the role of different levels of oxygen for normal cell function as well as control of complex signalling cascades is paramount to effectively build 3D tissues in vitro and their subsequent survival when implanted.
Acquisition of genome information from single-celled unculturable organisms (radiolaria) by exploiting genome profiling (GP)
Mariko Kouduka, Atsushi Matsuoka, Koichi Nishigaki
BMC Genomics , 2006, DOI: 10.1186/1471-2164-7-135
Abstract: Dendrogram was drawn regarding the single-celled unculturable species based on the similarity score termed PaSS, offering a consistent result with the conventional taxonomy of them built up based on phenotypes. This fact has shown that genome profiling-based technology developed here can obtain genome information being sufficient for identifying and classifying species from a single-celled organism.Since this method is so simple, general, and yet powerful, it can be applied to various organisms and cells, especially single-celled, uncluturable ones, for their genome analysis.Even in the post-genomic era, genome information is often difficult to obtain. This is especially true for organisms that are difficult to collect due to the extremes of their living environment. Radiolaria are protozoa used in the study of geology and environmental and ecological sciences because they leave a fossil of hard skeletal material that can provide a record of more than 500 million years. Studies on Radiolaria have progressed since the early work by Haeckel [1,2], which was based on phenotypic analysis. The diverse shapes of their skeletons have been useful characteristics for the identification of species. However, there is a need to obtain genomic information about these organisms in order to more fully understand their diversity and phylogeny. Recently, the gene encoding a small subunit of ribosomal RNA (18S rDNA) has been used for classifying species of Radiolaria, which are taxonomically controversial [3-5]. This is by no means an easy task not only because of the difficulty in collecting samples, but also the additional complication that each organism is composed of only a single cell, and methods for culturing Radiolaria in the laboratory are not available. This means that only a single set of genomic DNA molecules can be obtained for experiments such as PCR. Species identification and/or classification from a single cell has an ultimate value for various purposes: genome analy
Physics of Transport and Traffic Phenomena in Biology: from molecular motors and cells to organisms  [PDF]
Debashish Chowdhury,Andreas Schadschneider,Katsuhiro Nishinari
Physics , 2005, DOI: 10.1016/j.plrev.2005.09.001
Abstract: Traffic-like collective movements are observed at almost all levels of biological systems. Molecular motor proteins like, for example, kinesin and dynein, which are the vehicles of almost all intra-cellular transport in eukayotic cells, sometimes encounter traffic jam that manifests as a disease of the organism. Similarly, traffic jam of collagenase MMP-1, which moves on the collagen fibrils of the extracellular matrix of vertebrates, has also been observed in recent experiments. Traffic-like movements of social insects like ants and termites on trails are, perhaps, more familiar in our everyday life. Experimental, theoretical and computational investigations in the last few years have led to a deeper understanding of the generic or common physical principles involved in these phenomena. In particular, some of the methods of non-equilibrium statistical mechanics, pioneered almost a hundred years ago by Einstein, Langevin and others, turned out to be powerful theoretical tools for quantitaive analysis of models of these traffic-like collective phenomena as these systems are intrinsically far from equilibrium. In this review we critically examine the current status of our understanding, expose the limitations of the existing methods, mention open challenging questions and speculate on the possible future directions of research in this interdisciplinary area where physics meets not only chemistry and biology but also (nano-)technology.
Feasibility of Whole RNA Sequencing from Single-Cell mRNA Amplification  [PDF]
Yunbo Xu,Hongliang Hu,Jie Zheng,Biaoru Li
Genetics Research International , 2013, DOI: 10.1155/2013/724124
Abstract: Single-cell sampling with RNA-seq analysis plays an important role in reference laboratory; cytogenomic diagnosis for specimens on glass-slides or rare cells in circulating blood for tumor and genetic diseases; measurement of sensitivity and specificity in tumor-tissue genomic analysis with mixed-cells; mechanism analysis of differentiation and proliferation of cancer stem cell for academic purpose. Our single- cell RNA-seq technique shows that fragments were 250–450?bp after fragmentation, amplification, and adapter addition. There were 11.6 million reads mapped in raw sequencing reads (19.6 million). The numbers of mapped genes, mapped transcripts, and mapped exons were 31,332, 41,210, and 85,786, respectively. All QC results demonstrated that RNA-seq techniques could be used for single-cell genomic performance. Analysis of the mapped genes showed that the number of genes mapped by RNA-seq (6767 genes) was much higher than that of differential display (288 libraries) among similar specimens which we have developed and published. The single-cell RNA-seq can detect gene splicing using different subtype TGF-beta analysis. The results from using Q-rtPCR tests demonstrated that sensitivity is 76% and specificity is 55% from single-cell RNA-seq technique with some gene expression missing (2/8 genes). However, it will be feasible to use RNA-seq techniques to contribute to genomic medicine at single-cell level. 1. Introduction Clinical specimens are tremendously different from biological specimens in that the former contain mixed cells while the latter are mostly composed of pure cells. A mixed cell population in clinical samples can mask real results of genomic data, resulting in an inaccuracy of routine clinical genomic analysis and clinical genomic diagnosis. However, genomic medicine requires precise genomic profiling of clinical specimens to work for a clinical genomic diagnosis and to design personalized therapy for genetic and cancerous diseases. Like most routine diagnosis techniques [1, 2], clinical genomic analysis and genomic diagnosis techniques also have two prerequisites, that is, sensitivity and specificity, for clinical analysis and diagnosis [3–5]. In order to meet the requirements, two techniques can be considered: quantitative real-time PCR (Q-rtPCR) [6] and single-cell genomic analysis. After clinical genomic data, such as microarray data, is analyzed, Q-rtPCR is employed to support the microarray results by using similar primer design in the PCR as microarray probes [7]. Although Q-rtPCR is often used to confirm genomic data analysis as a
Proinflammatory effect in whole blood by free soluble bacterial components released from planktonic and biofilm cells
Jan Oscarsson, Maribasappa Karched, Bernard Thay, Casey Chen, Sirkka Asikainen
BMC Microbiology , 2008, DOI: 10.1186/1471-2180-8-206
Abstract: By employing an ex vivo insert model (filter pore size 20 nm) we demonstrated that the A. actinomycetemcomitans strain D7S and its derivatives, in both planktonic and in biofilm life-form, released free-soluble surface material independent of outer membrane vesicles. This material clearly enhanced the production of several proinflammatory cytokines (IL-1β, TNF-α, IL-6, IL-8, MIP-1β) in human whole blood, as evidenced by using a cytokine antibody array and dissociation-enhanced-lanthanide-fluorescent-immunoassay. In agreement with this, quantitative real-time PCR indicated a concomitant increase in transcription of each of these cytokine genes. Experiments in which the LPS activity was blocked with polymyxin B showed that the stimulatory effect was only partly LPS-dependent, suggesting the involvement of additional free-soluble factors. Consistent with this, MALDI-TOF-MS and immunoblotting revealed release of GroEL-like protein in free-soluble form. Conversely, the immunomodulatory toxins, cytolethal distending toxin and leukotoxin, and peptidoglycan-associated lipoprotein, appeared to be less important, as evidenced by studying strain D7S cdt/ltx double, and pal single mutants. In addition to A. actinomycetemcomitans a non-oral species, Escherichia coli strain IHE3034, tested in the same ex vivo model also released free-soluble surface material with proinflammatory activity.A. actinomycetemcomitans, grown in biofilm and planktonic form, releases free-soluble surface material independent of outer membrane vesicles, which induces proinflammatory responses in human whole blood. Our findings therefore suggest that release of surface components from live bacterial cells could constitute a mechanism for systemic stimulation and be of particular importance in chronic localized infections, such as periodontitis.Periodontitis is one of the most common chronic infections in humans, in which overgrowth of subgingival Gram-negative bacteria leads to chronic inflammation and gra
Lysis of a Single Cyanobacterium for Whole Genome?Amplification  [PDF]
Eric W. Hall,Samuel Kim,Visham Appadoo,Richard N. Zare
Micromachines , 2013, DOI: 10.3390/mi4030321
Abstract: Bacterial species from natural environments, exhibiting a great degree of genetic diversity that has yet to be characterized, pose a specific challenge to whole genome amplification (WGA) from single cells. A major challenge is establishing an effective, compatible, and controlled lysis protocol. We present a novel lysis protocol that can be used to extract genomic information from a single cyanobacterium of Synechocystis sp. PCC 6803 known to have multilayer cell wall structures that resist conventional lysis methods. Simple but effective strategies for releasing genomic DNA from captured cells while retaining cellular identities for single-cell analysis are presented. Successful sequencing of genetic elements from single-cell amplicons prepared by multiple displacement amplification (MDA) is demonstrated for selected genes (15 loci nearly equally spaced throughout the main chromosome).
Decontamination of MDA Reagents for Single Cell Whole Genome Amplification  [PDF]
Tanja Woyke, Alexander Sczyrba, Janey Lee, Christian Rinke, Damon Tighe, Scott Clingenpeel, Rex Malmstrom, Ramunas Stepanauskas, Jan-Fang Cheng
PLOS ONE , 2011, DOI: 10.1371/journal.pone.0026161
Abstract: Single cell genomics is a powerful and increasingly popular tool for studying the genetic make-up of uncultured microbes. A key challenge for successful single cell sequencing and analysis is the removal of exogenous DNA from whole genome amplification reagents. We found that UV irradiation of the multiple displacement amplification (MDA) reagents, including the Phi29 polymerase and random hexamer primers, effectively eliminates the amplification of contaminating DNA. The methodology is quick, simple, and highly effective, thus significantly improving whole genome amplification from single cells.
Modulation of mediators derived from whole blood or monocytic cells stimulated with lipopolysaccharide reduces endothelial cell activation
A Schildberger, T Stoifl, D Falkenhagen, V Weber
Critical Care , 2012, DOI: 10.1186/cc10619
Abstract: Whole blood or THP-1 cells (1 × 106 cells per ml medium containing 10% human plasma) [1] were stimulated with 10 ng/ml LPS from Escherichia coli for 4 hours. Mediator modulation was performed with either a specific adsorbent for TNFα which was based on sepharose particles functionalized with anti-TNFα antibodies, or with a selective albumin-coated polystyrene divinylbenzene copolymer (PS-DVB) [2]. Human umbilical vein endothelial cell (HUVEC) activation was monitored for 15 hours by measuring secretion of IL-6 and IL-8, as well as surface expression of the adhesion molecules ICAM-1 and E-selectin.Conditioned media derived from whole blood (CMB) or THP-1 cells (CMT) both contained approximately 1,300 pg/ml TNFα which is known to be an important stimulator for HUVEC [1,2]. However, CMB led to a significantly higher HUVEC activation as compared to CMT, as indicated by increased secretion of IL-6 and IL-8 (IL-6: 52,000 vs. 2,000 pg/ml; IL-8: 295,000 vs. 43,000 pg/ml), as well as significantly increased E-selectin surface expression (50 vs. 12 mean fluorescence intensity for CMP and CMT, respectively). Adsorption of inflammatory mediators from the conditioned medium of whole blood or THP-1 cells either with the specific TNFα adsorbent or with the selective PS-DVB beads resulted in decreased endothelial cell activation, as shown by statistically significant reduction of IL-6 and IL-8 secretion from HUVEC, as well as statistically significant reduction of surface expression of the adhesion molecules ICAM-1 and E-selectin. The reduction of HUVEC activation was more pronounced when applying the selective adsorbent showing that the modulation of more than one cytokine is more effective than removing TNFα alone.Inflammatory mediator modulation with specific or selective adsorbents reduces endothelial cell activation and thus may support the development of new therapies for sepsis.
Nanoliter Reactors Improve Multiple Displacement Amplification of Genomes from Single Cells  [PDF]
Yann Marcy,Thomas Ishoey,Roger S Lasken,Timothy B Stockwell,Brian P Walenz,Aaron L Halpern,Karen Y Beeson,Susanne M. D Goldberg,Stephen R Quake
PLOS Genetics , 2007, DOI: 10.1371/journal.pgen.0030155
Abstract: Since only a small fraction of environmental bacteria are amenable to laboratory culture, there is great interest in genomic sequencing directly from single cells. Sufficient DNA for sequencing can be obtained from one cell by the Multiple Displacement Amplification (MDA) method, thereby eliminating the need to develop culture methods. Here we used a microfluidic device to isolate individual Escherichia coli and amplify genomic DNA by MDA in 60-nl reactions. Our results confirm a report that reduced MDA reaction volume lowers nonspecific synthesis that can result from contaminant DNA templates and unfavourable interaction between primers. The quality of the genome amplification was assessed by qPCR and compared favourably to single-cell amplifications performed in standard 50-μl volumes. Amplification bias was greatly reduced in nanoliter volumes, thereby providing a more even representation of all sequences. Single-cell amplicons from both microliter and nanoliter volumes provided high-quality sequence data by high-throughput pyrosequencing, thereby demonstrating a straightforward route to sequencing genomes from single cells.
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