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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 mixture of cells sampled from a complex microbial ecosystem is introduced into the chip. Single cells are selected using an optical trap, and are sorted into chambers for cell lysis and genome amplification. Genomes are amplified in nanolitre MDA reactions to produce larger quantities of DNA (shown are SYBR Green–stained products in microfluidic reaction chambers). Sequencing libraries are created from the amplified genomic DNA for sequencing on a high‐throughput DNA sequencer. The sequence reads are assembled to recover the genome sequence, which is annotated to identify genes and pathways present in the original cell. Reprinted by permission from Macmillan Publishers Ltd: Nature Methods (Kalisky and Quake, 2011), copyright 2011. The microfluidics image was reprinted from Leslie (2011).
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f2: A mixture of cells sampled from a complex microbial ecosystem is introduced into the chip. Single cells are selected using an optical trap, and are sorted into chambers for cell lysis and genome amplification. Genomes are amplified in nanolitre MDA reactions to produce larger quantities of DNA (shown are SYBR Green–stained products in microfluidic reaction chambers). Sequencing libraries are created from the amplified genomic DNA for sequencing on a high‐throughput DNA sequencer. The sequence reads are assembled to recover the genome sequence, which is annotated to identify genes and pathways present in the original cell. Reprinted by permission from Macmillan Publishers Ltd: Nature Methods (Kalisky and Quake, 2011), copyright 2011. The microfluidics image was reprinted from Leslie (2011).

Mentions: An overview of an MDA set‐up using a microfluidic device is shown in Fig. 2, although FACS‐based methods are also often reported in literature (Rodrigue et al., 2009; Siegl and Hentschel, 2010). All DNA in the initial sample will be amplified, which renders the method very prone to DNA contamination. Another disadvantage of the initial method is uneven amplification of the genome, which results in high‐coverage sequencing of the amplified genomic regions while remaining sequences may not be sufficiently covered (Zhang et al., 2006). Marcy et al. (2007a) demonstrated that reducing MDA reaction volumes lowers non‐specific synthesis from contaminant DNA templates and unfavourable interactions between primers. The work of Rodrigue et al. (2009) demonstrated a biochemical method to normalize the products obtained in MDA reactions. They also discussed the problem of chimera formation linking non‐contiguous chromosomal regions in MDA (Dean et al., 2001; Zhang et al., 2006), which may hamper sequence assembly and render mate‐pair data less efficient in contig positioning. Several other single‐cell techniques are described in recent reviews by Wang and Bodovitz (2010), Kalisky and Quake (2011), and Pan et al. (2011). As data analysis from single‐cell amplified genomes is equally challenging, the software framework SmashCell has been developed to automate the main steps in sequence assembly, gene prediction, annotation and visualization (Harrington et al., 2010).


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

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

A mixture of cells sampled from a complex microbial ecosystem is introduced into the chip. Single cells are selected using an optical trap, and are sorted into chambers for cell lysis and genome amplification. Genomes are amplified in nanolitre MDA reactions to produce larger quantities of DNA (shown are SYBR Green–stained products in microfluidic reaction chambers). Sequencing libraries are created from the amplified genomic DNA for sequencing on a high‐throughput DNA sequencer. The sequence reads are assembled to recover the genome sequence, which is annotated to identify genes and pathways present in the original cell. Reprinted by permission from Macmillan Publishers Ltd: Nature Methods (Kalisky and Quake, 2011), copyright 2011. The microfluidics image was reprinted from Leslie (2011).
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3815255&req=5

f2: A mixture of cells sampled from a complex microbial ecosystem is introduced into the chip. Single cells are selected using an optical trap, and are sorted into chambers for cell lysis and genome amplification. Genomes are amplified in nanolitre MDA reactions to produce larger quantities of DNA (shown are SYBR Green–stained products in microfluidic reaction chambers). Sequencing libraries are created from the amplified genomic DNA for sequencing on a high‐throughput DNA sequencer. The sequence reads are assembled to recover the genome sequence, which is annotated to identify genes and pathways present in the original cell. Reprinted by permission from Macmillan Publishers Ltd: Nature Methods (Kalisky and Quake, 2011), copyright 2011. The microfluidics image was reprinted from Leslie (2011).
Mentions: An overview of an MDA set‐up using a microfluidic device is shown in Fig. 2, although FACS‐based methods are also often reported in literature (Rodrigue et al., 2009; Siegl and Hentschel, 2010). All DNA in the initial sample will be amplified, which renders the method very prone to DNA contamination. Another disadvantage of the initial method is uneven amplification of the genome, which results in high‐coverage sequencing of the amplified genomic regions while remaining sequences may not be sufficiently covered (Zhang et al., 2006). Marcy et al. (2007a) demonstrated that reducing MDA reaction volumes lowers non‐specific synthesis from contaminant DNA templates and unfavourable interactions between primers. The work of Rodrigue et al. (2009) demonstrated a biochemical method to normalize the products obtained in MDA reactions. They also discussed the problem of chimera formation linking non‐contiguous chromosomal regions in MDA (Dean et al., 2001; Zhang et al., 2006), which may hamper sequence assembly and render mate‐pair data less efficient in contig positioning. Several other single‐cell techniques are described in recent reviews by Wang and Bodovitz (2010), Kalisky and Quake (2011), and Pan et al. (2011). As data analysis from single‐cell amplified genomes is equally challenging, the software framework SmashCell has been developed to automate the main steps in sequence assembly, gene prediction, annotation and visualization (Harrington et al., 2010).

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