Generation and Screening of an Insertion Sequencing-Compatible Mutant Library of Campylobacter jejuni

The advent of next-generation sequencing technology has enabled experimental approaches to characterize large, complex populations of DNA molecules with high resolution. Included among these are methods to assess populations of transposon insertion libraries for the fitness cost of any particular mutant allele after applying selection to a population. These approaches have proven invaluable for identifying genetic factors that influence survival of bacterial pathogens within different environments, including animal hosts. One such method, termed insertion-site sequencing (INSeq), was designed to generate a 16 bp fragment of transposon-flanking genomic DNA captured during the protocol, which then serves as the substrate for massively parallel sequencing. Here we describe the generation of a transposon mutant library of Campylobacter jejuni amenable to INSeq and its use in identifying colonization determinants in a day-of-hatch chicken colonization model.