2015 Annual Science Report
University of Colorado, Boulder Reporting | JAN 2015 – DEC 2015
Advances in Gene Sequencing From Low-Biomass Water-Rock Hosted Ecosystems
One of the approaches our team is taking to explore rock-powered life is to study microorganisms hosted within rocks that are undergoing potentially life-supporting reactions with water. The chemistry of the rock microenvironments shapes the abundance, diversity and distribution of microbial life. In turn, that microbial life locally affects the in-situ geochemistry. This project is currently focusing on the successful extraction and sequencing of the exceedingly small amounts of DNA that accumulates within rocks, in order to successfully detect and characterize the rock-hosted life. Ultimately our improved approaches will support the application of next-generation DNA sequencing technology in the study of natural microbial ecosystems that are key for understanding the mechanisms of rock-powered life.
The rocky habitats that are of interest to the Rock-Powered Life team present many unique challenges to microbiologists. First, the total amount of biomass in deep-sea oceanic crust, for example, can be exceedingly low, challenging our ability to even detect life, not to mention our ability to characterize that life in any detail. Furthermore, many of the minerals that make up these rocks reduce the yield of DNA extractions, prevent effective DNA purification, and act as inhibitors to DNA sequencing reactions. The mineral properties of the rocks that we are studying diminish the efficiency of typical DNA-based techniques, compounding the problem of low biomass and the small amounts of DNA available in the rocks.
The Brazelton, Schrenk, and Spear labs have extensive experience in preparing DNA from minerals, and we have been adapting and optimizing existing DNA extraction and purification methodologies in order to improve our abilities to sequence DNA from low-biomass rocky habitats. For example, we are adapting SCODA (synchronous coefficient of drag attenuation) technology as implemented in the Aurora purification system (Boreal Genomics) to separate DNA molecules from minerals in an iterative process such that tiny quantities of DNA can be efficiently accumulated and concentrated in a single final preparation from multiple aliquots of large volumes of crushed rock. This will allow us to obtain the most possible DNA sequence information from rocks where the biomass may be isolated in small, difficult-to-detect pockets.
In addition to optimizing procedures for sequencing of total environmental DNA, we are also pursuing strategies for whole-genome sequencing of individual cells of bacteria and archaea collected from rocky habitats. This approach allows us to study rock-powered ecosystems at single-cell resolution, which is more biologically satisfying than working with random shotgun DNA sequences. Obtaining high-quality preparations of intact, single cells from minerals, however, is challenging, and we are currently adapting existing methods to meet these challenges. For example, our new approach prioritizes gentle handling of the cells in order to minimize destruction of the very few, precious cells that are typically available from rock samples. We are also pursuing spectroscopic analyses of the cells prior to sequencing in order to gain information about the physiological state of the individual cells and other phenotypes before harvesting each cell for whole-genome sequencing. This approach remains highly preliminary, but if successful, it will enable the linkage of genotype and phenotype at single-cell resolution, which is a kind of holy grail for microbiology.
Finally, we are exploring novel sequence data analysis techniques that will enable more efficient identification of distribution patterns of individual DNA sequences through large sequence datasets. The most immediate application of the approach will be the identification and removal of likely contaminant sequences based on their abundance patterns in existing datasets. This outcome will be crucial for proper interpretation of sequence data from low-biomass rock samples of the sort that are being studied by the Rock-Powered Life team because such samples are highly susceptible to contamination from many different sources that have many orders of magnitude higher biomass. If this contamination-tracing technique can be generalized, it could be developed into a tool for studying disperal of microorganisms in any kind of habitat by identifying sinks and sources of individual species through space and time.
PROJECT INVESTIGATORS:William Brazelton
PROJECT MEMBERS:Eric Boyd
RELATED OBJECTIVES:Objective 3.2
Origins and evolution of functional biomolecules
Earth's early biosphere.
Environment-dependent, molecular evolution in microorganisms
Co-evolution of microbial communities
Biochemical adaptation to extreme environments
Effects of environmental changes on microbial ecosystems
Biosignatures to be sought in nearby planetary systems