Generating Enriched Metagenomes from Active Microorganisms with DNA Stable Isotope Probing

Abstract

Cultivation-dependent methods have long been used to characterise the physiology of microorganisms after they have been isolated from the environment. In contrast, cultivation-independent methods help better identify the microorganisms that regulate specific biogeochemical processes in situ, regardless of their ability to grow in the laboratory. In this chapter, we describe how the cultivation-independent technique of DNA stable isotope probing (DNA-SIP) can be combined with targeted functional gene probing and metagenomic analyses to characterise the metabolic potential of active microbes from environmental samples, including those that assimilate labelled hydrocarbons. DNA-SIP enables the separation of DNA from microbes that have assimilated a given 13C-labelled substrate into their biomass (i.e., heavy DNA) from those that have not (i.e., light DNA). The heavy DNA can be used subsequently for PCR amplicon sequencing and metagenome sequencing, which can potentially lead to retrieving genomes of uncultivated and active microbial representatives.