Dataset Information

The Gut Microbiotassay: a high-throughput qPCR approach combinable with next generation sequencing to study gut microbial diversity.

ABSTRACT: BACKGROUND: The intestinal microbiota is a complex and diverse ecosystem that plays a significant role in maintaining the health and well-being of the mammalian host. During the last decade focus has increased on the importance of intestinal bacteria. Several molecular methods can be applied to describe the composition of the microbiota. This study used a new approach, the Gut Microbiotassay: an assembly of 24 primer sets targeting the main phyla and taxonomically related subgroups of the intestinal microbiota, to be used with the high-throughput qPCR chip 'Access Array 48.48', AA48.48, (Fluidigm®) followed by next generation sequencing. Primers were designed if necessary and all primer sets were screened against DNA extracted from pure cultures of 15 representative bacterial species. Subsequently the setup was tested on DNA extracted from small and large intestinal content from piglets with and without diarrhoea. The PCR amplicons from the 2304 reaction chambers were harvested from the AA48.48, purified, and sequenced using 454-technology. RESULTS: The Gut Microbiotassay was able to detect significant differences in the quantity and composition of the microbiota according to gut sections and diarrhoeic status. 454-sequencing confirmed the specificity of the primer sets. Diarrhoea was associated with a reduced number of members from the genus Streptococcus, and in particular S. alactolyticus. CONCLUSION: The Gut Microbiotassay provides fast and affordable high-throughput quantification of the bacterial composition in many samples and enables further descriptive taxonomic information if combined with 454-sequencing.

SUBMITTER: Hermann-Bank ML

PROVIDER: S-EPMC3879714 | biostudies-literature | 2013

REPOSITORIES: biostudies-literature

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The Gut Microbiotassay: a high-throughput qPCR approach combinable with next generation sequencing to study gut microbial diversity.

Hermann-Bank Marie Louise ML Skovgaard Kerstin K Stockmarr Anders A Larsen Niels N Mølbak Lars L

BMC genomics 20131114

<h4>Background</h4>The intestinal microbiota is a complex and diverse ecosystem that plays a significant role in maintaining the health and well-being of the mammalian host. During the last decade focus has increased on the importance of intestinal bacteria. Several molecular methods can be applied to describe the composition of the microbiota. This study used a new approach, the Gut Microbiotassay: an assembly of 24 primer sets targeting the main phyla and taxonomically related subgroups of the ...[more]

PMID: 24225361

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Project description:Amplicon sequencing has revolutionized our ability to study DNA collected from environmental samples by providing a rapid and sensitive technique for microbial community analysis that eliminates the challenges associated with lab cultivation and taxonomic identification through microscopy. In water resources management, it can be especially useful to evaluate ecosystem shifts in response to natural and anthropogenic landscape disturbances to signal potential water quality concerns, such as the detection of toxic cyanobacteria or pathogenic bacteria. Amplicon sequencing data consist of discrete counts of sequence reads, the sum of which is the library size. Groups of samples typically have different library sizes that are not representative of biological variation; library size normalization is required to meaningfully compare diversity between them. Rarefaction is a widely used normalization technique that involves the random subsampling of sequences from the initial sample library to a selected normalized library size. This process is often dismissed as statistically invalid because subsampling effectively discards a portion of the observed sequences, yet it remains prevalent in practice and the suitability of rarefying, relative to many other normalization approaches, for diversity analysis has been argued. Here, repeated rarefying is proposed as a tool to normalize library sizes for diversity analyses. This enables (i) proportionate representation of all observed sequences and (ii) characterization of the random variation introduced to diversity analyses by rarefying to a smaller library size shared by all samples. While many deterministic data transformations are not tailored to produce equal library sizes, repeatedly rarefying reflects the probabilistic process by which amplicon sequencing data are obtained as a representation of the amplified source microbial community. Specifically, it evaluates which data might have been obtained if a particular sample's library size had been smaller and allows graphical representation of the effects of this library size normalization process upon diversity analysis results.

Dataset Information

The Gut Microbiotassay: a high-throughput qPCR approach combinable with next generation sequencing to study gut microbial diversity.

Publications

The Gut Microbiotassay: a high-throughput qPCR approach combinable with next generation sequencing to study gut microbial diversity.

Similar Datasets

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