Species-by-Species Dissection of Microbiomes using Phage Display and Flow Sorting

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DESCRIPTION (provided by applicant): Metagenomics is a new scientific discipline that has developed in the last several years. It is both a set of research techniques, comprising many related approaches and methods, and a research field. As a scientific field, metagenomics attempts to resolve four tiers of questions: 1) what micro-organisms are present in a particular complex microbiome, such as human gut? 2) in what proportions? 3) what they are doing? and 4) how will they react to environmental changes, such as a change in diet? Currently, the approach to answer these questions has been one of brute force shotgun sequencing and 16S sequence surveys. However these technologies can only hint as to which kinds of bacteria are present, and the information provided tends to be biased to the commonest species. The majority of bacteria in complex microbiomes cannot presently be cultured and sequenced in a conventional way. Whole genome amplification (WGA) from single cells has been used in several studies. However, in the best cases, only 60% of a genome can be covered with the DNA obtained with WGA from a single cell. Studies have showed that the bias from WGA is random and coverage can be improved by adding more copies of the same genome. We propose here to change the metagenomics paradigm: rather than extracting all DNA in bulk without any independent information on the species that comprise it, we propose to develop tools to be able to analyze species one by one. This will be carried out by using phage display to select antibodies that recognize species in the population, and then to use such selected antibodies to characterize the abundance of the species by flow cytometry, purify it, and if necessary deplete the population of the species in order to repeat the process. The purified bacteria will be used as starting material for whole genome amplification, species characterization by rRNA analysis, and sequencing, if necessary. The antibodies developed within this proposal will be used to carry out the analyses indicated. Those developed within the context of the analysis of the human gut microbiome will also be very useful within the context of clinical studies in which bacterial composition may play an etiological role. An artificial bacterial mixture of E. coli and several other bacterial species will be used at the first stage of method development, and the microbiota in human gut will be analyzed in the later portion of the project. PUBLIC HEALTH RELEVANCE: Humans exist in collaboration with the bacteria that live within them, most of which are benign. However, it has recently been shown that the composition of the microbiome can have profound effects on the health of individuals. By developing new tools to analyze human microbiomes, we will provide additional methods to study and characterize different bacteria and elucidate their role in human disease.