Mapping transposon insertion sites within bacterial genomes by direct Sanger sequencing

Microbial biomedical research frequently involves mutagenesis by insertion of transposons into the genome. Currently, transposon insertion locations are typically elucidated by Next Generation Sequencing or by Sanger sequencing of PCR products. Here, transposons were located by direct Sanger sequencing of bacterial genomic DNA from both Salmonella enterica (Gram-negative) and Staphylococcus aureus (Gram-positive) cultures. DNA was prepared by shearing to a modal size of ∼2 kb followed by purification by paramagnetic beads. Sequencing reactions involved relatively minor modifications of standard protocols (e.g., extra sequencing polymerase, 75-100 PCR cycles); completed reactions were cleaned by ethanol-EDTA precipitation. Reads were generated on the ABI 3130xl Genetic Analyzer using 50-cm capillary arrays and a run protocol modified for extra sample injection time. Good quality reads of ∼500-800 nt were routinely generated; BLAST results returned nearly 100% matches to genomes in the NCBI database. As implemented, the optimized protocol (post-DNA extraction) could be performed within an 8-h workday (with sequencing results the following day) for ∼$10 (USD) per sequencing reaction. Although this method was developed to locate transposons inserted into bacterial genomes, it seems likely that it can be extended to generate sequence data from even native single-copy genes from small genomes (e.g., <5 Mb).