Development of a Sequence Capture Approach for Monitoring in situ Nitrogen Cycling Potential in Microbial Communities

Microorganisms play key roles in environmental functioning and understanding their responses to environmental perturbation is critical in developing accurate climate change models, a need highlighted by the DOE. However, their phylogenetic and functional diversity, abundance in the environment, and lack of cultured status make them challenging to study. While there are several molecular methods available that have been extremely important in advancing our knowledge of microbial ecology, there are serious drawbacks to each of the available methods. This Phase I project aims to develop a sequence capture approach for detecting and monitoring the functional potential of microbial communities to take advantage of the strengths of microarray and sequencing methods while minimizing their weaknesses. The sequence capture approach will allow for the monitoring of numerous functional genes at one time while providing a mechanism to confirm sequence identity and detect new sequence information. In Phase I, capture probes for the nitrogen-fixing gene, nifH, will be designed using two typical approaches and available sequences from public databases. Hybridization conditions will be optimized for this functional gene and the method will be tested on real-world samples. In later Phases, the gene coverage will be expanded to cover a wide range of functional genes and conditions will be optimized for detection of multiple genes and for use with RNA. This method will greatly expand the ability of researchers to examine the functional capability of microbial communities, increase the number of available sequences, and provide greater insight into microbial community responses.