Mapping transposon insertion sites within bacterial genomes by direct Sanger sequencing.

Area of Science:

• Microbiology
• Genomics
• Molecular Biology

Background:

• Transposon mutagenesis is crucial in microbial biomedical research.
• Current methods for identifying transposon insertion sites, like Next Generation Sequencing and PCR-Sanger sequencing, can be time-consuming and costly.

Purpose of the Study:

• To develop and optimize a direct Sanger sequencing protocol for efficient and cost-effective identification of transposon insertion locations in bacterial genomes.
• To demonstrate the applicability of this method to both Gram-negative (Salmonella enterica) and Gram-positive (Staphylococcus aureus) bacteria.

Main Methods:

• Bacterial genomic DNA was sheared to ~2 kb and purified using paramagnetic beads.
• Direct Sanger sequencing was performed with minor protocol modifications, including increased sequencing polymerase and 75-100 PCR cycles.
• Sequencing was conducted on an ABI 3130xl Genetic Analyzer, generating ~500-800 nt reads.

Main Results:

• The optimized protocol successfully located transposons in both Salmonella enterica and Staphylococcus aureus.
• High-quality reads with nearly 100% BLAST matches to NCBI genomes were routinely obtained.
• The method, post-DNA extraction, can be completed within an 8-hour workday for approximately $10 per reaction.

Conclusions:

• This direct Sanger sequencing approach provides a rapid, economical, and efficient alternative for determining transposon insertion sites in bacterial genomes.
• The protocol's adaptability suggests potential for sequencing native single-copy genes in small genomes (<5 Mb).