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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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A schematic overview of poribacterial metabolism as deduced from SAG sequencing. Reprinted by permission from Macmillan Publishers Ltd: The ISME Journal (Siegl et al., 2011), copyright 2011.
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f4: A schematic overview of poribacterial metabolism as deduced from SAG sequencing. Reprinted by permission from Macmillan Publishers Ltd: The ISME Journal (Siegl et al., 2011), copyright 2011.

Mentions: Siegl et al. (2011) 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. With the finding of a second putative PKS system showing high similarity to the lipopolysaccharide type I PKS WcbR from Nitrosomonas and Burkholderia, as well as RkpA from Sinorhizobium fredii, they suggested that Poribacteria contain at least two different types of PKS systems and their products may be involved in sponge–microbe interactions. This study showed that single‐cell genomics is highly capable of dissecting the genomic information from unculturable bacteria, shedding light on genomic organization, metabolic functions and possibly new insight in the debate on the origin of sponge bioactive compounds.


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

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

A schematic overview of poribacterial metabolism as deduced from SAG sequencing. Reprinted by permission from Macmillan Publishers Ltd: The ISME Journal (Siegl et al., 2011), copyright 2011.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3815255&req=5

f4: A schematic overview of poribacterial metabolism as deduced from SAG sequencing. Reprinted by permission from Macmillan Publishers Ltd: The ISME Journal (Siegl et al., 2011), copyright 2011.
Mentions: Siegl et al. (2011) 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. With the finding of a second putative PKS system showing high similarity to the lipopolysaccharide type I PKS WcbR from Nitrosomonas and Burkholderia, as well as RkpA from Sinorhizobium fredii, they suggested that Poribacteria contain at least two different types of PKS systems and their products may be involved in sponge–microbe interactions. This study showed that single‐cell genomics is highly capable of dissecting the genomic information from unculturable bacteria, shedding light on genomic organization, metabolic functions and possibly new insight in the debate on the origin of sponge bioactive compounds.

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.

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