Sequence-verified two-allele transposon mutant library for Pseudomonas aeruginosa PAO1

Abstract

Mutant hunts using comprehensive sequence-defined libraries make it possible to identify virtually all of the nonessential functions required for different bacterial processes. However, the success of such screening depends on the accuracy of mutant identification in the mutant library used. To provide a high-quality library for Pseudomonas aeruginosa PAO1, we created a sequence-verified collection of 9,437 transposon mutants that provides genome coverage and includes two mutants for most genes. Mutants were cherry-picked from a larger library, colony-purified, and resequenced both individually using Sanger sequencing and in a pool using Tn-seq. About 8% of the insertion assignments were corrected, and in the final library nearly 93% of the transposon locations were confirmed by at least one of the resequencing procedures. The extensive sequence verification and inclusion of more than one mutant for most genes should help minimize missed or erroneous genotype-phenotype assignments in studies using the new library.

Fig 1

Generation and sequence verification of the two-allele library. The steps followed in creating and checking the makeup of the library are indicated.

Fig 2

Distribution of insertions in a representative region of the genome. Insertions in a 13-kbp region of the genome are shown with their verification levels indicated. More than half of the locations were confirmed by both Sanger sequencing and Tn-seq.