Experimental and analytical considerations for randomly barcoded transposon insertion sequencing (RB-TnSeq) studies

Randomly barcoded transposon insertion sequencing (RB-TnSeq) is an efficient, multiplexed method to determine microbial gene function during growth under a selection condition of interest. This technique applies to growth, tolerance, and persistence studies in a variety of hosts, but the wealth of data generated can add to the difficulty in identifying the most critical gene hits for a given condition. Experimental and analytical methods to improve the resolution of RB-TnSeq are proposed in the accompanying work, using Pseudomonas putida KT2440 as an example organism. Baseline selection was analyzed by comparing fitness results from a M9 + 20 mM D-glucose enrichment inoculated from baseline (T0) LB cultures washed once in M9 salts or T0 cultures prepared in M9 + 20 mM D-glucose. Additionally, utility of a medium reference was analyzed by comparing fitness in M9 with 20 mM D-glucose (Reference) to M9 with 10 mM ferulate (enrichment), both inoculated from M9 + 20 mM D-glucose T0 cultures, as a means to delineate enrichment-specific outcomes (ferulate catabolism) from general media outcomes (growth in M9, generally). Finally, these datasets were used to assess the value in trimming out transposon count data from transposons located as gene termini, prior to performing fitness calculations. These considerations provide practitioners with several options to improve the resolution of RB-TnSeq outputs, enabling accurate characterization of the organism of interest.