VI Environmental risk assessment of the deliberate release of engineered micro-organisms

Next generation sequencing-based metagenomics for monitoring soil microbiota

Hana Yi, Department of Environmental Health, Korea University and Jongsik Chun, School of Biological Sciences, Seoul National University

DNA sequencing is a powerful method to unravel the genetic diversity of micro-organisms in nature. In recent years, revolutionary next-generation sequencing technologies have become widely used in various microbiological disciplines, including microbial taxonomy and ecology. This chapter reviews the species concept of prokaryotes, including bacteria and Archaea, and presents the development of a comprehensive methodology for monitoring microbes in soil. Next-generation sequencing-enabled metagenomics should be useful and can be widely applied to modern microbiology and biotechnology.

Next-generation sequencing

In 1977, the chain-termination based DNA sequencing method was developed by Frederick Sanger (Sanger et al., 1977). The principle of this chain-termination method (or Sanger method) was the incorporation of dideoxynucleotide triphosphates (ddNTPs) as DNA chain terminators during the synthesis of complementary strand of template single-stranded DNA. As the ddNTPs are radioactively labelled, DNA fragments that are the result of chain termination after incorporation of ddNTPs can be detected based on one-dimensional polyacrylamide gel electrophoresis and autoradiography. The dramatic improvement of the original Sanger method was achieved by using fluorescently labelled ddNTPs and capillary electrophoresis (Smith et al., 1985; 1986). By the development of this automated Sanger sequencing method, DNA sequencing has become easier and orders of magnitude faster. The partially automated Sanger DNA sequencing method has dominated the fields of molecular biology for almost two decades and led to numerous scientific accomplishments, including the completion of the only finished-grade human genome sequence (Consortium, 2004). Despite substantial technical improvements during this period of time, the limitations of automated Sanger sequencing arose and presented a strong need for new and improved technologies for DNA sequencing with much higher throughput, such as required for sequencing large numbers of human genomes. Recent efforts have been directed towards the development of methods with a completely new basis, leaving Sanger sequencing with fewer reported incremental advances (Metzker, 2010).

Very recently, several types of high-throughput and low-cost platform for DNA sequencing methods have been developed and have made important progress in DNA sequencing (Mardis, 2008; Margulies et al., 2005; Valouev et al., 2008). The automated Sanger method is considered as a “first-generation” technology, and these newer methods are referred to as next-generation sequencing (NGS) (Pettersson et al., 2009). Currently, several NGS technologies are commercially available or about to become available, including Roche/454 (Margulies et al., 2005), Illumina/Solexa (Bentley et al., 2008), Life Technologies/APG (Valouev et al., 2008), Helicos BioSciences (Harris et al., 2008), Polonator (Shendure et al., 2005), Pacific Biosciences (Eid et al., 2009), Oxford Nanopore Technologies (Clarke et al., 2009) and Life Technologies/Ion Torrent (Rothberg et al., 2011). These new technologies employ various strategies applying multiple technological disciplines and rely on a combination of template preparation, sequencing and imaging, and genome alignment and assembly methods. One of the major advances offered by NGS is the ability to generate an enormous volume of data cheaply - in some cases in excess of 1 billion short reads per instrument run. This feature puts NGS into the new realm of experimentation such as transcriptomics, beyond just determining the order of bases (Metzker, 2010).

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