AbstractCommensal bacteria from the human intestinal microbiota play important roles in health and disease. Research into the mechanisms by which these bacteria exert their effects is hampered by the complexity of the microbiota and by the strict growth requirements of the individual species. The assembly of ordered transposon insertion libraries, in which nearly all nonessential genes have been disrupted and the strains stored as independent monocultures, would be a transformative resource for

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... research into many microbiota members. However, assembly of these libraries must be fast and inexpensive in order to empower investigation of the large number of species that typically compose gut communities. The methods used to generate ordered libraries must also be adapted to the anaerobic growth requirements of most intestinal bacteria. We have developed a protocol to assemble ordered libraries of transposon insertion mutants that is fast, cheap, and effective for even strict anaerobes. The protocol differs from currently available methods by making use of cell sorting to order the library and barcoded transposons to facilitate the localization of ordered mutations in the library. By tracking transposon insertions using barcode sequencing, our approach increases the accuracy and reduces the time and effort required to locate mutants in the library. Ordered libraries can be sorted and characterized over the course of two weeks using this approach. We expect this protocol will lower the barrier to generating comprehensive, ordered mutant libraries for many species in the human microbiota, allowing for new investigations into genotype-phenotype relationships within this important microbial ecosystem.