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Chapter 15 : Single-Cell Genomics

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Single-Cell Genomics, Page 1 of 2

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Abstract:

Large metagenomic studies demonstrate the central theme, the microbial heterogeneity of individual microbial cells, which will drive single-cell genomics and microbiology. This chapter gives an overview of the current state of the art and highlights the different technological steps necessary to achieve the total genome sequence from a single microbial cell. It addresses (i) methods to isolate single bacterial cells, (ii) DNA isolation and amplification from a single microorganisms, (iii) DNA sequencing, and (iv) applications and future outlook. Many applications of single-cell genomics require careful DNA amplification to overcome several problems and potential limitations. Multiple displacement amplification (MDA) amplifies all DNA present within the sample, including contaminating DNAs, multicopy sequences, and plasmids, thus making sequence annotation more challenging. MDA techniques are also being used in microbial ecology for strategies beyond single-cell genomics applications, which should accelerate their refinement and adoption. The current and potential uses of MDA warrant further work on addressing the potential biases and problems with its application. One of the most promising new sequencing technologies is the 454 sequencing platforms. Continued improvements in DNA sequencing techniques, bioinformatics, and data analysis over the past few years have helped reduce the cost and time associated with sequencing a genome.

Citation: Keller M, Schadt C, Palumbo A. 2008. Single-Cell Genomics, p 267-278. In Zengler K (ed), Accessing Uncultivated Microorganisms. ASM Press, Washington, DC. doi: 10.1128/9781555815509.ch15
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FIGURE 1

Schematic of DNA amplification through MDA. Bacterial cells will be lysed followed by DNA amplification through MDA using the strand displacement enzyme Phi 29.

Citation: Keller M, Schadt C, Palumbo A. 2008. Single-Cell Genomics, p 267-278. In Zengler K (ed), Accessing Uncultivated Microorganisms. ASM Press, Washington, DC. doi: 10.1128/9781555815509.ch15
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Image of FIGURE 2
FIGURE 2

Targeted access to microbial cells directly from the environment. This approach combines high specificity derived from the stringent hybridization of oligonucleotide probes to target rRNA in a taxonomically predefined cellular population, with high sensitivity and throughput from the detection and separation of labeled cells from complex mixtures of organisms generated by flow cytometry. The isolated cells are then subject to WGA MDA, followed by DNA sequencing.

Citation: Keller M, Schadt C, Palumbo A. 2008. Single-Cell Genomics, p 267-278. In Zengler K (ed), Accessing Uncultivated Microorganisms. ASM Press, Washington, DC. doi: 10.1128/9781555815509.ch15
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