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Simultaneous quantification of multiple bacteria by the BactoChip microarray designed to target species-specific marker genes.

Ballarini A, Segata N, Huttenhower C, Jousson O - PLoS ONE (2013)

Bottom Line: The microarray successfully distinguished among bacterial species from 21 different genera using 60-mer probes targeting a novel set of in silico identified high-resolution marker genes.The BactoChip additionally proved accurate in determining species-level relative abundances over a 100-fold dynamic range in complex bacterial communities and with a low limit of detection (0.1%).In combination with the continually increasing number of sequenced bacterial genomes, future iterations of the technology could enable to highly accurate clinically-oriented tools for rapid assessment of bacterial community composition and relative abundances.

View Article: PubMed Central - PubMed

Affiliation: Centre for Integrative Biology, University of Trento, Trento, Italy. ballarini@science.unitn.it

ABSTRACT
Bacteria are ubiquitous throughout the environment, the most abundant inhabitants of the healthy human microbiome, and causal pathogens in a variety of diseases. Their identification in disease is often an essential step in rapid diagnosis and targeted intervention, particularly in clinical settings. At present, clinical bacterial detection and discrimination is primarily culture-based, requiring both time and microbiological expertise, especially for bacteria that are not easily cultivated. Higher-throughput molecular methods based on PCR amplification or, recently, microarrays are reaching the clinic as well. However, these methods are currently restricted to a small set of microbes or based on conserved phylogenetic markers such as the 16S rRNA gene, which are difficult to resolve at the species or strain levels. Here, we designed and experimentally validated the BactoChip, an oligonucleotide microarray for bacterial detection and quantification. The chip allows the culture-independent identification of bacterial species, also determining their relative abundances in complex communities as occur in the commensal microbiota or in clinical settings. The microarray successfully distinguished among bacterial species from 21 different genera using 60-mer probes targeting a novel set of in silico identified high-resolution marker genes. The BactoChip additionally proved accurate in determining species-level relative abundances over a 100-fold dynamic range in complex bacterial communities and with a low limit of detection (0.1%). In combination with the continually increasing number of sequenced bacterial genomes, future iterations of the technology could enable to highly accurate clinically-oriented tools for rapid assessment of bacterial community composition and relative abundances.

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Design of the BactoChip and its application for microbial species identification and quantification.(A) Schematic overview of computational design and its output. (A1) Complete genomes from 186 bacterial species were retrieved from the National Centre for Biotechnology Information microbial database. (A2) Gene sequences core to each target species were defined on the basis of sequence conservation within each clade. Red dots represent the distribution of core genes shared by strains within and outside a target clade. (A3) Core genes unique to each target species were selected by sequence alignment against all available archaeal and bacterial sequences. (A4) Oligonucleotide probes were designed for up to 10 identified unique genes for each target bacterial species. Each probe color represents specificity to a defined bacterial species. (B) Experimental design and example data. (B1) DNA from microbial communities was tested on the BactoChip. Green dots represent Cy3 bound to genomic DNA fragments from a sample hybridized to the chip. (B2) Species relative abundances are finally inferred by normalization of the fluorescence signal for each probe and species.
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pone-0055764-g001: Design of the BactoChip and its application for microbial species identification and quantification.(A) Schematic overview of computational design and its output. (A1) Complete genomes from 186 bacterial species were retrieved from the National Centre for Biotechnology Information microbial database. (A2) Gene sequences core to each target species were defined on the basis of sequence conservation within each clade. Red dots represent the distribution of core genes shared by strains within and outside a target clade. (A3) Core genes unique to each target species were selected by sequence alignment against all available archaeal and bacterial sequences. (A4) Oligonucleotide probes were designed for up to 10 identified unique genes for each target bacterial species. Each probe color represents specificity to a defined bacterial species. (B) Experimental design and example data. (B1) DNA from microbial communities was tested on the BactoChip. Green dots represent Cy3 bound to genomic DNA fragments from a sample hybridized to the chip. (B2) Species relative abundances are finally inferred by normalization of the fluorescence signal for each probe and species.

Mentions: We developed an oligonucleotide microarray to simultaneously identify a broad range of bacterial species with clinical relevance to human health. In contrast to current phylogenetic microarray approaches for microbial identification, which target individual universally conserved genes such as the 16S rRNA, we identified in silico those genes that most unequivocally characterized each bacterial species. Unique, conserved regions within these genes were subsequently targeted for microarray probe design (Figure 1).


Simultaneous quantification of multiple bacteria by the BactoChip microarray designed to target species-specific marker genes.

Ballarini A, Segata N, Huttenhower C, Jousson O - PLoS ONE (2013)

Design of the BactoChip and its application for microbial species identification and quantification.(A) Schematic overview of computational design and its output. (A1) Complete genomes from 186 bacterial species were retrieved from the National Centre for Biotechnology Information microbial database. (A2) Gene sequences core to each target species were defined on the basis of sequence conservation within each clade. Red dots represent the distribution of core genes shared by strains within and outside a target clade. (A3) Core genes unique to each target species were selected by sequence alignment against all available archaeal and bacterial sequences. (A4) Oligonucleotide probes were designed for up to 10 identified unique genes for each target bacterial species. Each probe color represents specificity to a defined bacterial species. (B) Experimental design and example data. (B1) DNA from microbial communities was tested on the BactoChip. Green dots represent Cy3 bound to genomic DNA fragments from a sample hybridized to the chip. (B2) Species relative abundances are finally inferred by normalization of the fluorescence signal for each probe and species.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3569451&req=5

pone-0055764-g001: Design of the BactoChip and its application for microbial species identification and quantification.(A) Schematic overview of computational design and its output. (A1) Complete genomes from 186 bacterial species were retrieved from the National Centre for Biotechnology Information microbial database. (A2) Gene sequences core to each target species were defined on the basis of sequence conservation within each clade. Red dots represent the distribution of core genes shared by strains within and outside a target clade. (A3) Core genes unique to each target species were selected by sequence alignment against all available archaeal and bacterial sequences. (A4) Oligonucleotide probes were designed for up to 10 identified unique genes for each target bacterial species. Each probe color represents specificity to a defined bacterial species. (B) Experimental design and example data. (B1) DNA from microbial communities was tested on the BactoChip. Green dots represent Cy3 bound to genomic DNA fragments from a sample hybridized to the chip. (B2) Species relative abundances are finally inferred by normalization of the fluorescence signal for each probe and species.
Mentions: We developed an oligonucleotide microarray to simultaneously identify a broad range of bacterial species with clinical relevance to human health. In contrast to current phylogenetic microarray approaches for microbial identification, which target individual universally conserved genes such as the 16S rRNA, we identified in silico those genes that most unequivocally characterized each bacterial species. Unique, conserved regions within these genes were subsequently targeted for microarray probe design (Figure 1).

Bottom Line: The microarray successfully distinguished among bacterial species from 21 different genera using 60-mer probes targeting a novel set of in silico identified high-resolution marker genes.The BactoChip additionally proved accurate in determining species-level relative abundances over a 100-fold dynamic range in complex bacterial communities and with a low limit of detection (0.1%).In combination with the continually increasing number of sequenced bacterial genomes, future iterations of the technology could enable to highly accurate clinically-oriented tools for rapid assessment of bacterial community composition and relative abundances.

View Article: PubMed Central - PubMed

Affiliation: Centre for Integrative Biology, University of Trento, Trento, Italy. ballarini@science.unitn.it

ABSTRACT
Bacteria are ubiquitous throughout the environment, the most abundant inhabitants of the healthy human microbiome, and causal pathogens in a variety of diseases. Their identification in disease is often an essential step in rapid diagnosis and targeted intervention, particularly in clinical settings. At present, clinical bacterial detection and discrimination is primarily culture-based, requiring both time and microbiological expertise, especially for bacteria that are not easily cultivated. Higher-throughput molecular methods based on PCR amplification or, recently, microarrays are reaching the clinic as well. However, these methods are currently restricted to a small set of microbes or based on conserved phylogenetic markers such as the 16S rRNA gene, which are difficult to resolve at the species or strain levels. Here, we designed and experimentally validated the BactoChip, an oligonucleotide microarray for bacterial detection and quantification. The chip allows the culture-independent identification of bacterial species, also determining their relative abundances in complex communities as occur in the commensal microbiota or in clinical settings. The microarray successfully distinguished among bacterial species from 21 different genera using 60-mer probes targeting a novel set of in silico identified high-resolution marker genes. The BactoChip additionally proved accurate in determining species-level relative abundances over a 100-fold dynamic range in complex bacterial communities and with a low limit of detection (0.1%). In combination with the continually increasing number of sequenced bacterial genomes, future iterations of the technology could enable to highly accurate clinically-oriented tools for rapid assessment of bacterial community composition and relative abundances.

Show MeSH
Related in: MedlinePlus