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PROJECT TOPIC:  METAGENOMIC ANALYSIS OF BACTERIA POPULATION IN A HYDROCARBON PULLUTED WATER BODIES.
Department:  Microbiology
AMOUNT:  15,000
FORMAT:   MS WORD
PAGES:  88
 
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ABSTRACT

The emergence of metagenomics-based approaches in biology has overcome historical culture-based biases in microbiological studies. This has also enabled a more comprehensive assessment of the microbial ecology of environmental samples. The subsequent development of next-generation sequencing technologies, able to produce hundreds of millions of sequences at improved cost and speed, necessitated a computational shift from user-supervised alignment and analysis pipelines, that were used previously for vector-based metagenomic studies that relied on Sanger sequencing. Current computational advances have expanded the scope of microbial biogeography studies and offered novel insights into microbial responses to environmental variation and anthropogenic inputs into ecosystems. However, new biostatistical and computational approaches are required to handle the large volume and complexity of these new multivariate datasets. While this has allowed more complete characterization of taxonomic, phylogenetic and functional microbial diversity, these tools are still limited by methodological biases, incomplete databases, and the high cost of fully characterizing environmental biodiversity. This review addresses the evolution of methods to monitor surface waters and characterize environmental samples through the recent computational advances in metagenomics, with an emphasis on the study of surface waters. These new methods have provided an abundance of opportunities to expand our understanding of the interaction between microbial communities and public health. Specifically, they have allowed for comprehensive monitoring of bacterial communities in surface waters for changes in community structure associated with faecal contamination and the presence of human pathogens, rather than relying on only a few indicator bacteria to direct public health concerns.

Keywords: environmental samples, metagenomics, next-generation sequencing, 16S rDNA

 

 

 

 

 

 

 

 

INTRODUCTION

Microorganisms can be found across all environments. Adequate sample collection is the initial and essential step to achieve a comprehensive coverage of the microbial diversity. For aquatic studies, the collection of large volumes of water followed by filtration is recommended since it increases the chance of retrieving rare groups (Brito et al,2016). After sample collection, an optional enrichment culture step can be performed to maximize the abundance of a targeted group of microorganisms by providing its ideal growth conditions. Further screening for specific phenotypic features may be performed during the cultivation step in order to target microbial species of unusual metabolism. Genetic material can be obtained from samples by several methods that include physical and/or chemical cell lysis followed by extraction and purification of nucleic acids (DNA or RNA). However, the amount of non-biological matter associated with the biological material may interfere with the extraction, quantification and amplification processes. Thus, different samples will require different extraction methods as a good representation of the biological diversity relies on the efficiency of the nucleic acid extraction step. The extracted genetic material must be free of amplification inhibitors and special attention must be given when dealing with samples retrieved from polluted sites (Cardoso et al., 2010).

Metagenomics may shed light in understanding the complex degradation routes of xenobiotics, which is currently poorly understood. These compounds can be toxic for living organisms and represent a threat to ecosystems. The use of molecular techniques based on genomic analysis can be applied in the monitoring of enzymes associated with the metabolism of xenobiotics, including herbicides and other pollutants (Malik et al., 2008). Microbial communities can be screened by gene-specific PCR to detect the presence of genes of interest within a community. This method, however, has a bias of favoring previously known genes. An alternative method, which allows for the identification of novel genes and metabolic pathways, is the construction of expression libraries from metagenomic DNA. Through this method, positive clones expressing randomly cloned environmental genes are screened for their capacity to metabolize a specific substrate by plating in media containing a particular compound. In this way, the clones that metabolize this compound can be selected for DNA sequencing. By gene-specific PCR it is possible to quickly identify genes associated with biodegradation in an environmental sample and sometimes affiliate them with a specific taxonomic group. This approach is limited by the fact that the metabolic pathways to which xenobiotics are submitted can encompass several steps, requiring more than a single enzyme to be catabolized. Therefore, cloning the full set of genes would be required to obtain the desired phenotype, which is not always possible since those enzymes can be encoded by different genes spread throughout the genome. An alternative procedure for novel gene discovery is by Stable Isotope Probing (SIP). SIP is based on the incorporation of stable isotope-labeled substrates into molecular biomarkers. Once labeled substrates are provided to a microbial community, those microorganisms capable of metabolizing this substrate (e.g. a pollutant molecule) are likely to incorporate labeled atoms in their DNA, RNA and protein molecules. Nucleic acids with labeled atoms can be extracted to retrieve genomic material exclusively from a community capable of metabolizing a desired substrate (Malik et al., 2008). Furthermore, the sequencing of complete genomes of microorganisms can also reveal a set of metabolic genes with important applications for the biodegradation process, representing another important tool for bioremediation strategies.

 

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