Transposon sequencing (Tn-seq) is a powerful tool for functional genomics of bacteria. Tn-seq combines transposon mutagenesis with next generation sequencing to assess genetic requirements at a genome-wide scale and identify essential and conditionally essential genes. An efficient application of this experimental approach relies on robust protocols for transposon mutagenesis system and Tn-seq amplicon library preparation method. However, the existing approaches for the Tn-seq amplicon library preparation have several shortcomings. Hence, we present a robust, fast, specific, and cost-effective approach for the transposon mutagenesis of Salmonella Typhimurium and Tn-seq amplicon library preparation for Illumina sequencing. Besides S. Typhimurium that was used here for illustration, this protocol can also be used for other bacteria. In particular, the procedure for Tn-seq amplicon library preparation can be broadly applicable to any transposon elements. We delineate comprehensive step-by-step protocols for transposon mutagenesis and Tn-seq amplicon library such that it can be reproduced effortlessly by other researchers.
Keywords: Functional genomics; Illumina sequencing; Salmonella Typhimurium; Tn-seq amplicon library; Transposon mutagenesis.