Metagenomic Pyrosequencing and Microbial Identification

Department of Molecular Virology and Microbiology, Baylor College of Medicine, Houston, TX, USA.
Clinical Chemistry (Impact Factor: 7.91). 04/2009; 55(5):856-66. DOI: 10.1373/clinchem.2008.107565
Source: PubMed


The Human Microbiome Project has ushered in a new era for human metagenomics and high-throughput next-generation sequencing strategies.
This review describes evolving strategies in metagenomics, with a special emphasis on the core technology of DNA pyrosequencing. The challenges of microbial identification in the context of microbial populations are discussed. The development of next-generation pyrosequencing strategies and the technical hurdles confronting these methodologies are addressed. Bioinformatics-related topics include taxonomic systems, sequence databases, sequence-alignment tools, and classifiers. DNA sequencing based on 16S rRNA genes or entire genomes is summarized with respect to potential pyrosequencing applications.
Both the approach of 16S rDNA amplicon sequencing and the whole-genome sequencing approach may be useful for human metagenomics, and numerous bioinformatics tools are being deployed to tackle such vast amounts of microbiological sequence diversity. Metagenomics, or genetic studies of microbial communities, may ultimately contribute to a more comprehensive understanding of human health, disease susceptibilities, and the pathophysiology of infectious and immune-mediated diseases.

Download full-text


Available from: Ruth Ann Luna
  • Source
    • "Due to the inherent limitations of conventional methods, microbiologists typically focus on dominant microorganisms in complex microbial communities. However, the rapid development of next generation sequencing (NGS) technologies has laid the foundation for novel approaches in characterizing complex microbial consortia particularly with massively parallel DNA sequencing of short hypervariable regions of small subunit (SSU) which, make it possible to detect relatively low abundant microorganisms in a consortium (Sogin, et al., 2006; Huse, et al., 2008; Petrosino et al., 2009; Shokralla et al., 2012). NGS has not only increased the throughput of analysis, but it has also lowered the cost per sequence read (Kircher and Kelso, 2010). "
    [Show abstract] [Hide abstract]
    ABSTRACT: Characterization of commercial microbial consortia products for human and environmental health risk assessment is a major challenge for regulatory agencies. As a means to develop an approach to assess the potential environmental risk of these products, research was conducted to compare four genomics methods for characterizing bacterial communities; (i) Denaturing Gradient Gel Electrophoresis (DGGE), (ii) Clonal- Restriction Fragment Length Polymorphism (C/RFLP), (iii) partial 16S rDNA amplification, cloning followed by Sanger sequencing (PRACS) and (iv) Next-Generation Sequencing (NGS) based on Ion Torrent technology. A commercially available microbial consortium, marketed as a remediation agent for degrading petroleum hydrocarbon contamination in soil and water, was assessed. The bacterial composition of the commercial microbial product was characterized using the above four methods. PCR amplification of 16S rDNA was performed targeting the variable region V6 for DGGE, C/RFLP and PRACS and V5 for Ion Torrent sequencing. Ion Torrent technology was shown to be a promising tool for initial screening by detecting the majority of bacteria in the consortium that were also detected by DGGE, C/RFLP and PRACS. Additionally, Ion Torrent sequencing detected some of the bacteria that were claimed to be in the product, while three other methods failed to detect these specific bacteria. However, the relative proportions of the microbial composition detected by Ion Torrent were found to be different from DGGE, C/RFLP and PRACS, which gave comparable results across these three methods. The discrepancy of the Ion Torrent results may be due to the short read length generated by this technique and the targeting of different variable regions on the 16S rRNA gene used in this study. Arcobacter spp. a potential pathogenic bacteria was detected in the product by all methods, which was further confirmed using genus and species-specific PCR, RFLP and DNA-based sequence analyses. However, the viability of Arcobacter spp. was not confirmed. This study suggests that a combination of two or more methods may be required to ascertain the microbial constituents of a commercial microbial consortium reliably and for the presence of potentially human pathogenic contaminants. Copyright © 2014. Published by Elsevier B.V.
    Full-text · Article · Dec 2014 · Journal of Microbiological Methods
  • Source
    • "The recent development of next generation sequencing (NGS) technologies has renewed the interest of using microorganisms as tools to control vector populations or parasite and virus transmission (Boissière et al., 2012; Metzker, 2010; Minard et al., 2013; Petrosino et al., 2009; Ricci et al., 2012; Sogin et al., 2006). "
    [Show abstract] [Hide abstract]
    ABSTRACT: During their immature life stages, malaria mosquitoes are exposed to a wide array of microbes and contaminants from the aquatic habitats. Although prior studies have suggested that environmental exposure shapes the microbial community structure in the adult mosquito, most reports have focused on laboratory-based experiments and on a single mosquito epithelium, the gut. In this study, we investigated the influence of the breeding site on the development of the Anopheles coluzzii and Anopheles gambiae microbiota in natural conditions. We characterized bacterial communities from aquatic habitats, at surface microlayer and subsurface water levels, to freshly emerge adult mosquitoes using multiplexed 16S rRNA gene pyrosequencing and we separately analyzed the microbiota associated with the different epithelia of adult individual, midguts, ovaries and salivary glands. We found that the distribution of bacterial communities in the aquatic habitats differed according to the depth of water collections. Inter-individual variation of bacterial composition was large in larvae guts but adult mosquitoes from a same breeding site shared quite similar microbiota. Although some differences in bacterial abundances were highlighted between the different epithelia of freshly emerged An. coluzzii and An. gambiae, an intriguing feature from our study is the particular similarity of the overall bacterial communities. Our results call for further investigations on the bacterial population dynamics in the different tissues to determine the distinctive characteristics of each microbiota during the mosquito lifespan and to identify specific interactions between certain key phyla or species and the insect life history traits.
    Full-text · Article · Oct 2014 · Infection Genetics and Evolution
    • "Enthusiasm for the metagenomic approach runs high. Its theoretical promise to reach all the microorganisms in an ecosystem thanks to the small pool of DNA (and RNA) extracted from the ecosystem inhabitants along with the potential of sequencing technologies to provide the genetic map of millions of microorganism genomes without any need for their cultivation have already offered a deep glimpse into microbial comprehension (Eisen, 2007; Petrosino et al., 2010; Riesenfeld et al., 2004). However, after twenty years of metagenomic development, the soil remains an ecosystem defying detailed analysis and understanding. "
    [Show abstract] [Hide abstract]
    ABSTRACT: A culture independent approach was developed for recovering individual bacterial cells out of communities from complex environments including soils and sediments where autofluorescent contaminants hinder the use of fluorescence based techniques. For that purpose fifty nanometer sized streptavidin-coated superparamagnetic nanoparticles were used to chemically bond biotin-functionalized plasmid DNA molecules. We show that micromagnets can efficiently trap magnetically labeled transformed Escherichia coli cells after these bacteria were subjected to electro-transformation by these nanoparticle-labeled plasmids. Among other applications, this method could extend the range of approaches developed to study DNA dissemination among environmental bacteria without requiring cultivability of recombinant strains or expression of heterologous genes in the new hosts.
    No preview · Article · Sep 2014 · Journal of Microbiological Methods
Show more