High-Throughput Next Generation Sequencing pp 25-33

Part of the Methods in Molecular Biology book series (MIMB, volume 733)

Whole-Genome Sequencing of Unculturable Bacterium Using Whole-Genome Amplification

Protocol

Abstract

More than 99% of microorganisms on the earth are unculturable with known culturing techniques. The emergence of metagenomics with high-throughput sequencing technologies has enabled researchers to capture a comprehensive view of a complex bacterial community which comprises both culturable and unculturable species. However, the function of an individual species remains difficult to elucidate in a conventional metagenomic study, which generates numerous genomic fragments of unidentifiable origins at a species or genus level. This limitation hampers any in-depth investigations of the community and its unculturable bacterial members. Recently, as an alternative or compensatory approach, genomics targeting a single unculturable bacterial species in a complex community has been proposed. In this approach, whole-genome amplification technique using Phi29 DNA polymerase is applied to obtain a sufficient quantity of DNA for genome sequence analysis from only a single to a thousand bacterial cells. It is expected that a combination of the conventional metagenomics and this single-species-targeting genomics provides a great progress in understanding of the ecology, physiology, and evolution of unculturable microbial communities.

Key words

Whole-genome amplification Phi29 DNA polymerase Pyrosequence Uncultivable Uncultured Environmental genomics Metagenomics Single-cell genomics Termite Symbiosis 

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Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  1. 1.Department of Biological Sciences, Graduate School of Bioscience and BiotechnologyTokyo Institute of TechnologyTokyoJapan

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