Extended Data Fig. 1: TraDIS library generation and sequencing workflow and predicted outcome of transposon insertions in tsi (immunity) genes in each library background.

a, En masse transposon (Tn) mutagenesis in T6SS active (PAKΔretS) or T6SS inactive (PAKΔretSΔH1) backgrounds was performed to generate pooled transposon mutant libraries of ~2 million mutants each. These libraries were then separately passaged overnight at high contact density and the genomic DNA from recovered mutants was harvested. This genomic DNA was then fragmented and adaptors ligated to each end prior to PCR enrichment for transposon-containing DNA fragments. The pooled DNA population was then subjected to TraDIS DNA sequencing. b, Artemis (http://www.sanger.ac.uk/science/tools/artemis - version 17/0.1) plot file showing distribution of transposon insertions (red and blue lines correspond to insertions mapped from either forward or reverse sequence reads) in immunity gene (tsi2 in this case) in the T6SS active library background (top panel - no insertions permitted) and in the T6SS inactive library background (right - insertions are permitted). The other H1-T6SS immunity genes detected, as well as the putative previously unidentified T6SS immunity genes (Table 1) had a similar distribution of transposon insertions in each library background as for tsi2. Panel (a) adapted from Barquist et al., (2013)⁵⁶.