library_construction_protocol: Qiagen Genomic-tip 100/G kit ~2 μg of genomic DNA was fragmented to a size of ~300 base-pairs using a Covaris Focused ultrasonicator (Covaris), followed by end repair using the NEBNext End Repair module (NEB), A-tailing using the NEBNext dA-Tailing module (NEB), and adapter ligation using the NEBNext Quick Ligation Module with a custom splinkerette adaptor (SplA5). PCR enrichment of fragments containing transposon sequence was then carried out using transposon- and adaptor-specific primers using Kapa HiFi HotStart ReadyMix (Kapa Biosystems) for 10 to 20 cycles. PCR products were analyzed on an Agilent Bioanalyzer using a High Sensitivity DNA kit (Agilent Technologies), and by SYBR green qPCR (Kapa Biosystems). Sequencing was then performed on an Illumina HiSeq 2000 or MiSeq using transposon-specific primers and a custom dark-cycle sequencing protocol at the Wellcome Sanger Institute or the King Abdullah University of Science and Technology Bioscience Core Laboratory (Salmonella Typhimurium A130 and D23580). All custom adaptors, primers, and sequencing protocols are described in Barquist et al. "The TraDIS toolkit: sequencing and analysis for dense transposon mutant libraries" Bioinformatics 2016 and publicly available at  https://github.com/sanger-pathogens/Bio-Tradis/tree/master/recipes. transposon insertion sequencing