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

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., 2006; Appl. Environ. Microbiol. 72, 4329-4337). Previously, insertion sites have been determined for 5000 mutants of this library. Combining an adapter-free, inverse PCR method for sequencing library preparation with next generation sequencing, we identified 4473 novel insertion sites, increasing the total number of transposon mutants with known insertion site to 9562. The number of protein-coding genes that were hit at least once by a transposon increased by 1231 to a total number of 3673 disrupted genes, which represents 59% of the predicted protein-coding genes in S. meliloti.