We describe a deep-sequencing procedure for tracking large numbers of transposon 
mutants of Pseudomonas aeruginosa. The procedure employs a new Tn-seq 
methodology based on the generation and amplification of single-strand circles 
carrying transposon junction sequences (the Tn-seq circle method), a method 
which can be used with virtually any transposon. The procedure reliably 
identified more than 100,000 transposon insertions in a single experiment, 
providing near-saturation coverage of the genome. To test the effectiveness of 
the procedure for mutant identification, we screened for mutations reducing 
intrinsic resistance to the aminoglycoside antibiotic tobramycin. Intrinsic 
tobramycin resistance had been previously analyzed at genome scale using 
mutant-by-mutant screening and thus provided a benchmark for evaluating the new 
method. The new Tn-seq procedure identified 117 tobramycin resistance genes, the 
majority of which were then verified with individual mutants. The group of genes 
with the strongest mutant phenotypes included nearly all (13 of 14) of those 
with strong mutant phenotypes identified in the previous screening, as well as a 
nearly equal number of new genes. The results thus show the effectiveness of the 
Tn-seq method in defining the genetic basis of a complex resistance trait of P. 
aeruginosa and indicate that it can be used to analyze a variety of 
growth-related processes.