Metagenomic Pyrosequencing and Microbial Identification
ABSTRACT 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.
Full-textDOI: · Available from: Ruth Ann Luna, Aug 12, 2015
- SourceAvailable from: Izhar U H Khan
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- "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). "
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.Journal of Microbiological Methods 12/2014; 108. DOI:10.1016/j.mimet.2014.11.013 · 2.10 Impact Factor
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- "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). "
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.Infection Genetics and Evolution 10/2014; 28. DOI:10.1016/j.meegid.2014.09.029 · 3.26 Impact Factor
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- "We have observed a similar trend, i.e., in our 16S sequencing based analyses, L. kefiranofaciens abundance is reported to be significantly lower than the abundance we report using the WGS metagenomic analysis and closer to the ratios obtained in other studies. This difference is potentially due to the different primers used in amplifying certain regions of the 16S rRNA gene, which may limit the ability to assess the dominant species' abundance with high accuracy (Hong et al., 2009; Petrosino et al., 2009). "
ABSTRACT: Kefir grains as a probiotic have been subject to microbial community identification using culture-dependent and independent methods that target specific strains in the community, or that are based on limited 16S rRNA analysis. We performed whole genome shotgun pyrosequencing using two Turkish Kefir grains. Sequencing generated 3,682,455 high quality reads for a total of ∼1.6 Gbp of data assembled into 6151 contigs with a total length of ∼24 Mbp. Species identification mapped 88.16% and 93.81% of the reads rendering 4 Mpb of assembly that did not show any homology to known bacterial sequences. Identified communities in the two grains showed high concordance where Lactobacillus was the most abundant genus with a mapped abundance of 99.42% and 99.79%. This genus was dominantly represented by three species Lactobacillus kefiranofaciens, Lactobacillus buchneri and Lactobacillus helveticus with a total mapped abundance of 97.63% and 98.74%. We compared and verified our findings with 16S pyrosequencing and model based 16S data analysis. Our results suggest that microbial community profiling using whole genome shotgun data is feasible, can identify novel species data, and has the potential to generate a more accurate and detailed assessment of the underlying bacterial community, especially for low abundance species.Food Microbiology 08/2014; 41:42–51. DOI:10.1016/j.fm.2014.01.014 · 3.37 Impact Factor