Abstract
Transposon mutagenesis greatly facilitates the study of gene function in microorganisms ranging from viruses to fungi. Traditionally, one would study individual transposon mutants with interesting phenotypes one mutant at a time. Here, we describe methods for the study of tens of thousands of transposon mutants in parallel in the bacterial pathogen Vibrio cholerae using transposon-sequencing. The first section outlines methods for making a saturated transposon mutant library. The second section outlines methods for massively parallel sequencing of the transposon junctions. The third section outlines methods for analyzing the sequence data to calculate the fitness contribution of genes.