Abstract
Metagenomics has evolved over the last 3 decades from the analysis of single genes and their apparent diversity in an ecosystem to the provision of complex genetic information relating to whole ecosystems. Metagenomics is a vast subject area in terms of methodology, which encompasses a suite of molecular technologies employed to investigate genomic information from all members of a microbial community. However, the relatively recent developments in high-throughput sequencing platforms have meant that metagenomic can be performed simply by extracting DNA and sequencing it. Here, we outline explicit methodologies for the extraction of metagenomic DNA from marine and sediments/soil environmental samples, the subsequent production and sequencing of large-insert metagenomic libraries, and also shotgun pyrosequencing considerations. We also provide relevant advice on bioinformatic analyses of the complex metagenomic datasets. We hope that the information provided here will be useful to establish the techniques in most reasonably equipped molecular biology laboratories.
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References
Rusch DB, Halpern AL, Sutton G, Heidelberg KB, Williamson S, et al. (2007) The Sorcerer II Global Ocean Sampling expedition: northwest Atlantic through eastern tropical Pacific. PLoS Biol. 5, 398–  431
DeLong EF, Preston CM, Mincer T, Rich V, Hallam SJ, et al. (2006) Community genomics among stratified microbial assemblages in the ocean’s interior. Science 311, 496  –503.
Gilbert JA, Field D, Huang Y, Edwards R, Li W, et al. (2008) Detection of Large Numbers of Novel Sequences in the Metatranscriptomes of Complex Marine Microbial Communities. PLoS ONE 3(8): e3042. doi:10.1371/journal.pone.0003042
Temperton B, Field D, Oliver A, Tiwari B, Joint I, Gilbert JA (2009) Bias in assessments of marine microbial biodiversity in fosmid libraries as evaluated by pyrosequencing. ISME J. 3, 792–  6.
Smith CJ, Nedwell DB, Dong LF, Osborn AM (2007) Diversity and abundance of nitrate reductase genes (narG and napA), nitrite reductase genes (nirS and nrfA), and their transcripts in estuarine sediments. Appl. Environ. Microbiol. 73, 3612–3622.
Sambrook J, Fritsch EF, Maniatis T (2000) Molecular Cloning: a laboratory manual. 3rd ed. N.Y., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press.
Altschul, SF., Gish W, Miller W, Myers EW, Lipman DJ (1990) Basic local alignment search tool. J. Mol. Biol. 215, 403  –10.
Korf I, Yandell M, Bedell J (2003) BLAST. O’Reilly Media.
Gomez-Alvarez V, Teal TK, Schmidt TM, (2009) Systematic artifacts in metagenomes from complex microbial communities. ISME 3, 1314  –1317.
Quince C, Lanzan A, Curtis TP, Davenport RJ, Hall N, Head IM, Read IF, Sloan WT (2009) Accurate determination of microbial diversity from 454 pyrosequencing data. Nature Methods 6, 639–641.
Anderson MJ (2001) A new method for non-parametric multivariate analysis of variance. Austral Ecology 26, 32–  46
Clarke KR (1993) Non-parametric multiÂvariate analyses of changes in community structure. Australian Journal of Ecology 18, 117–143.
Acknowledgments
The author would like to thank Neil Hall from the NERC/University of Liverpool Advanced Genomics Facility.
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Gilbert, J.A., Laverock, B., Temperton, B., Thomas, S., Muhling, M., Hughes, M. (2011). Metagenomics. In: Kwon, Y., Ricke, S. (eds) High-Throughput Next Generation Sequencing. Methods in Molecular Biology, vol 733. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-61779-089-8_12
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DOI: https://doi.org/10.1007/978-1-61779-089-8_12
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Publisher Name: Humana Press, Totowa, NJ
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