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Single-cell genomics: unravelling the genomes of unculturable microorganisms.

de Jager V, Siezen RJ - Microb Biotechnol (2011)

View Article: PubMed Central - PubMed

Affiliation: Netherlands Bioinformatics Centre, Nijmegen, The Netherlands.

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Microbial genomics and related transcriptomics methods rely on culturing techniques to obtain enough DNA suitable for high‐throughput sequencing without resorting to DNA amplification techniques... A few microgram of DNA is needed for most common next‐generation sequencing methods... Metagenome or transcriptome analysis of microorganisms has been described for biofilms consisting of a single species by scraping of the biofilm to obtain enough material, but for multi‐species biofilms this method results in a metagenome or metatranscriptome dataset... Whereas classical next‐generation sequencing to determine an organism's genome sequence relies on pooling DNA from 10–10 cells, single‐cell genomics relies on whole‐genome amplification from a single cell... Other amplification techniques like random‐primed PCR result in a more over‐ and under‐representation of different regions of the template DNA and generate very short fragments... MDA, however, results in fragments of 12–100 kb rendering them suitable for sequencing... The remaining reads were assembled into a draft genome, misassemblies due to chimeras were corrected manually, and subsequent application of primer walking, sequencing PCR products and Illumina sequencing resulted in a final finished genome (Fig.  3). ) used FACS to isolate cells from the candidate phylum Poribacteria and subsequently MDA to obtain a SAG... These bacteria are almost exclusively found in marine sponges as symbionts and resist cultivation efforts... The SAG of 1.88 Mb was contained in 1597 contigs, which covered an estimated two‐thirds of the total genomic DNA based on the distribution of tRNA genes and their specificities found in the contigs... Nevertheless, a comprehensive overview of poribacterial metabolism could be deduced (Fig.  4)... The extensive Sup‐type polyketide synthases found in the SAG of Poribacteria confirmed the previously proposed assignment of Sup‐PKS to this species... Single‐cell metabolome and proteome/peptidome analyses are still in their infancy, as these compounds cannot be amplified and their analysis requires technological breakthroughs in pushing the limits of detection... Since the introduction of single‐cell genomics, there have been surprisingly few reports of successful reconstruction of whole genomes from single unculturable bacterial cells (Table 1)... In this light, adaptation of single‐cell genome sequencing using microfluidic approaches towards RNA‐seq transcriptome analysis of single cells using next‐generation mRNA sequencing should become increasingly important.

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Photograph of a single‐cell isolation and genome amplification chip capable of processing nine samples in parallel (eight cells, one positive control). A. To visualize the architecture, the channels and chambers have been filled with blue food colouring and the control lines to actuate the valves have been filled with red food colouring (scale bar 5 mm). B. Schematic diagram of the automated sorting procedure. Closed valves are shown in red, open valves are transparent. Cells are drawn in green. C. Typical result of cell sorting showing for each unit (seven with a single cell and one negative control without a cell) a colour combination of a phase contrast image (gray) and a fluorescence image (green). A green overlaid square has been placed around the cell to ease visualization, whereas a red crossed square indicates the absence of cell. Scale bar is 100 µm. Reprinted from Marcy et al. (2007a).
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f1: Photograph of a single‐cell isolation and genome amplification chip capable of processing nine samples in parallel (eight cells, one positive control). A. To visualize the architecture, the channels and chambers have been filled with blue food colouring and the control lines to actuate the valves have been filled with red food colouring (scale bar 5 mm). B. Schematic diagram of the automated sorting procedure. Closed valves are shown in red, open valves are transparent. Cells are drawn in green. C. Typical result of cell sorting showing for each unit (seven with a single cell and one negative control without a cell) a colour combination of a phase contrast image (gray) and a fluorescence image (green). A green overlaid square has been placed around the cell to ease visualization, whereas a red crossed square indicates the absence of cell. Scale bar is 100 µm. Reprinted from Marcy et al. (2007a).

Mentions: Several methods exist to extract and investigate single microbial cells from their environment. Flow cytometry or fluorescence‐activated cell sorting (FACS) has been used since the 1970s and its applications in microbiology were recognized early (Fouchet et al., 1993); recent advances are described by Müller and Nebe‐von‐Caron (2010), Wang and Bodovitz (2010), and Wang et al. (2010). Micromanipulation has been described by Kvist et al. (2007) and more recently by Woyke et al. (2010). Microfluidic device techniques are shown to be effective by combining the separation of cells and subsequently performing biochemical reactions on the device itself, thereby maximizing reaction yield (Marcy et al., 2007a) (Fig. 1).


Single-cell genomics: unravelling the genomes of unculturable microorganisms.

de Jager V, Siezen RJ - Microb Biotechnol (2011)

Photograph of a single‐cell isolation and genome amplification chip capable of processing nine samples in parallel (eight cells, one positive control). A. To visualize the architecture, the channels and chambers have been filled with blue food colouring and the control lines to actuate the valves have been filled with red food colouring (scale bar 5 mm). B. Schematic diagram of the automated sorting procedure. Closed valves are shown in red, open valves are transparent. Cells are drawn in green. C. Typical result of cell sorting showing for each unit (seven with a single cell and one negative control without a cell) a colour combination of a phase contrast image (gray) and a fluorescence image (green). A green overlaid square has been placed around the cell to ease visualization, whereas a red crossed square indicates the absence of cell. Scale bar is 100 µm. Reprinted from Marcy et al. (2007a).
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3815255&req=5

f1: Photograph of a single‐cell isolation and genome amplification chip capable of processing nine samples in parallel (eight cells, one positive control). A. To visualize the architecture, the channels and chambers have been filled with blue food colouring and the control lines to actuate the valves have been filled with red food colouring (scale bar 5 mm). B. Schematic diagram of the automated sorting procedure. Closed valves are shown in red, open valves are transparent. Cells are drawn in green. C. Typical result of cell sorting showing for each unit (seven with a single cell and one negative control without a cell) a colour combination of a phase contrast image (gray) and a fluorescence image (green). A green overlaid square has been placed around the cell to ease visualization, whereas a red crossed square indicates the absence of cell. Scale bar is 100 µm. Reprinted from Marcy et al. (2007a).
Mentions: Several methods exist to extract and investigate single microbial cells from their environment. Flow cytometry or fluorescence‐activated cell sorting (FACS) has been used since the 1970s and its applications in microbiology were recognized early (Fouchet et al., 1993); recent advances are described by Müller and Nebe‐von‐Caron (2010), Wang and Bodovitz (2010), and Wang et al. (2010). Micromanipulation has been described by Kvist et al. (2007) and more recently by Woyke et al. (2010). Microfluidic device techniques are shown to be effective by combining the separation of cells and subsequently performing biochemical reactions on the device itself, thereby maximizing reaction yield (Marcy et al., 2007a) (Fig. 1).

View Article: PubMed Central - PubMed

Affiliation: Netherlands Bioinformatics Centre, Nijmegen, The Netherlands.

AUTOMATICALLY GENERATED EXCERPT
Please rate it.

Microbial genomics and related transcriptomics methods rely on culturing techniques to obtain enough DNA suitable for high‐throughput sequencing without resorting to DNA amplification techniques... A few microgram of DNA is needed for most common next‐generation sequencing methods... Metagenome or transcriptome analysis of microorganisms has been described for biofilms consisting of a single species by scraping of the biofilm to obtain enough material, but for multi‐species biofilms this method results in a metagenome or metatranscriptome dataset... Whereas classical next‐generation sequencing to determine an organism's genome sequence relies on pooling DNA from 10–10 cells, single‐cell genomics relies on whole‐genome amplification from a single cell... Other amplification techniques like random‐primed PCR result in a more over‐ and under‐representation of different regions of the template DNA and generate very short fragments... MDA, however, results in fragments of 12–100 kb rendering them suitable for sequencing... The remaining reads were assembled into a draft genome, misassemblies due to chimeras were corrected manually, and subsequent application of primer walking, sequencing PCR products and Illumina sequencing resulted in a final finished genome (Fig.  3). ) used FACS to isolate cells from the candidate phylum Poribacteria and subsequently MDA to obtain a SAG... These bacteria are almost exclusively found in marine sponges as symbionts and resist cultivation efforts... The SAG of 1.88 Mb was contained in 1597 contigs, which covered an estimated two‐thirds of the total genomic DNA based on the distribution of tRNA genes and their specificities found in the contigs... Nevertheless, a comprehensive overview of poribacterial metabolism could be deduced (Fig.  4)... The extensive Sup‐type polyketide synthases found in the SAG of Poribacteria confirmed the previously proposed assignment of Sup‐PKS to this species... Single‐cell metabolome and proteome/peptidome analyses are still in their infancy, as these compounds cannot be amplified and their analysis requires technological breakthroughs in pushing the limits of detection... Since the introduction of single‐cell genomics, there have been surprisingly few reports of successful reconstruction of whole genomes from single unculturable bacterial cells (Table 1)... In this light, adaptation of single‐cell genome sequencing using microfluidic approaches towards RNA‐seq transcriptome analysis of single cells using next‐generation mRNA sequencing should become increasingly important.

Show MeSH
Related in: MedlinePlus