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Pyrosequence Analysis of Unamplified and Whole Genome Amplified DNA from Hydrocarbon-Contaminated Groundwater

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Abstract

Pyrosequence data was used to analyze the composition and metabolic potential of a metagenome from a hydrocarbon-contaminated site. Unamplified and whole genome amplified (WGA) sequence data was compared from this source. According to MG-RAST, an additional 2,742,252 bp of DNA was obtained with the WGA, indicating that WGA has the ability to generate a large amount of DNA from a small amount of starting sample. However, it was observed that WGA introduced a bias with respect to the distribution of the amplified DNA and the types of microbial populations that were accessed from the metagenome. The dominant order in the WGA metagenome was Flavobacteriales, whereas the unamplified metagenome was dominated by Actinomycetales as determined by RDPII and CARMA databases. According to the SEED database, the subsystems shown to be present for the individual metagenomes were associated with the metabolic potential that was expected to be present in the contaminated groundwater, such as the metabolism of aromatic compounds. A higher percentage (4.4) of genes associated with the metabolism of aromatic compounds was identified in the unamplified metagenome when compared to the WGA metagenome (0.66%). This could be attributed to the increased number of hydrocarbon degrading bacteria that had been accessed from this metagenome (Mycobacteria, Nocardia, Brevibacteria, Clavibacter, Rubrobacter, and Rhodoccocus). Therefore, it was possible to relate the taxonomic groups accessed to the contamination profile of the metagenome. By collating the sequencing data obtained pre- and post-amplification, this study provided insight regarding the survival strategies of microbial communities inhabiting contaminated environments.

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Abbreviations

CARMA:

CARMA is a software pipeline for the characterization of species composition and the genetic potential of microbial samples using short reads. In contrast to the traditional 16S-rRNA approach for taxonomical classification, CARMA uses reads that encode for known proteins. By assigning the taxonomic origins to each read, a profile is constructed which characterizes the taxonomic composition of the corresponding community

EGT (Expressed Gene Tag):

A unique stretch of DNA within a coding region of a gene that is useful for identifying full-length genes and serves as a landmark for mapping. An EGT is a sequence tagged site (STS) derived from cDNA

Metagenomics:

The genomic analysis of microorganisms by direct extraction and/or cloning of DNA from an assemblage of microorganisms (also refereed to as environmental and community genomics)

MGRAST:

MetaGenome Rapid Annotation Subsystems Technology MGRAST: is a fully automated service for annotating metagenome samples that provides annotation of sequence fragments, their phylogenetic classification, metabolic reconstructions and comparison tools

Pyrosequencing:

A method of DNA sequencing (determining the order of nucleotides in DNA) based on the “sequencing by synthesis” principle. It differs from Sanger sequencing, in that it relies on the detection of pyrophosphate release on nucleotide incorporation, rather than chain termination with dideoxynucleotides

RDPII:

The Ribosomal Database Project (RDP) provides ribosome related data and services to the scientific community, including online data analysis and aligned and annotated Bacterial and Archaeal small-subunit 16S rRNA sequences

Whole genome amplification:

An increasingly common technique through which minute amounts of DNA can be multiplied to generate quantities suitable for genetic testing and analysis

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Acknowledgments

The authors wish to thank the National Research Foundation for financial support and TIA (Dr James Sakwa and colleagues) for the pyrosequencing.

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Authors and Affiliations

  1. Discipline of Microbiology, Faculty of Science and Agriculture, University of KwaZulu-Natal (Westville Campus), Private Bag X54001, Durban, 4000, Republic of South Africa

    Nathlee S. Abbai, Algasan Govender, Rehana Shaik & Balakrishna Pillay

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  1. Nathlee S. Abbai
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  2. Algasan Govender
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  3. Rehana Shaik
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Correspondence to Balakrishna Pillay.

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Abbai, N.S., Govender, A., Shaik, R. et al. Pyrosequence Analysis of Unamplified and Whole Genome Amplified DNA from Hydrocarbon-Contaminated Groundwater. Mol Biotechnol 50, 39–48 (2012). https://doi.org/10.1007/s12033-011-9412-8

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