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Methods Enzymol. 2011;495:81-98. doi: 10.1016/B978-0-12-386905-0.00006-1.

Functional metagenomics of methylotrophs.

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1
Department of Microbiology, University of Washington, Seattle, Washington, USA.

Abstract

It is widely recognized that most microbes in the biosphere remain uncultured and unknown. In the recent few years, whole genome shotgun (WGS) sequencing of environmental DNA (metagenomics) has revolutionized the field of environmental microbiology by allowing one to tap into the genomic content of microbial communities in specific ecological niches, deducing information on their biochemical potentials. However, ascribing specific functions to specific organisms remains very difficult in most cases, due to low sequence coverage and the lack of sequence assembly that result from metagenomics of complex microbial communities. Therefore, methods that link specific biogeochemical processes to specific members of such complex natural communities are urgently needed. We have developed and implemented a functional metagenomics approach that allows such a connection via substrate-specific stable isotope labeling, followed by WGS sequencing of the labeled DNA to describe bacterial populations involved in metabolism of single-carbon compounds in a freshwater lake. We also developed a pipeline for community transcript analysis based on ultrashort read high-throughput sequencing of messenger RNA, matching these to a specific scaffold. The methodologies described in this chapter can be applied in a wide variety of ecosystems for targeting methylotrophs as well as other functional guilds of microbes.

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