Massive parallel insertion site sequencing of an arrayed Sinorhizobium meliloti signature-tagged mini-Tn 5 transposon mutant library.

Transposon mutagenesis in conjunction with identification of genomic transposon insertion sites is a powerful tool for gene function studies. We have implemented a protocol for parallel determination of transposon insertion sites by Illumina sequencing involving a hierarchical barcoding method that allowed for tracking back insertion sites to individual clones of an arrayed signature-tagged transposon mutant library. This protocol was applied to further characterize a signature-tagged mini-Tn 5 mutant library comprising about 12,000 mutants of the symbiotic nitrogen-fixing alphaproteobacterium Sinorhizobium meliloti (Pobigaylo et al.