(378d) Microdevices and Materials for Genomic Analysis of Single Microbial Cells

Next-generation sequencing technologies have made whole-genome sequencing of microbes routine, as long as the microbe can be grown in pure culture to provide sufficient quantities of clonal DNA for library preparation.  For microbes that are not cultured, which includes the vast majority of taxa both in the environment and within the human body, other techniques must be used to analyze microbial genomes.  Metagenomics is useful for analyzing genes and pathways at the level of microbial communities, but assembly of complete microbial genomes from a mixed population is difficult except for the simplest communities.  We have developed a suite of technologies that complement metagenomic techniques by analyzing the genomic content of single cells.  We begin with complex microbial communities, isolate individual cells, and then amplify to prepare sequencing libraries.  Key aspects of the technology include: (1) compartmentalization of single cells in nanoliter-volume droplets, as ideal "nanoreactors" for amplifying the genome of a single microbial cell, (2) multiple displacement amplification (MDA) using Phi29 DNA polymerase, with special consideration to purity requirements when amplifying single cells, and (3) labeling and sorting techniques based on fluorescence in situ hybridization (FISH), either before or after amplification, to identify interesting or novel microbes based on phylogeny (i.e. 16S rRNA sequence) or presence of functional genes.  Our pipeline utilizes a combination of conventional instrumentation (FACS) and novel microfluidics, with the ultimate goal of producing sequencing libraries from single-amplified genomes (SAGs), with increased ability to target rare or novel microbes relative to "blind" sorting techniques.