IN-Seq/Tn-Seq
IN-Seq/Tn-Seq
Transposon sequencing (Tn-Seq) or insertion sequencing (INSeq) accurately determines quantitative genetic interactions. In this method, a transposon with flanking Mmel digestion sites is transposed into bacteria which, after culturing, can help detect the frequency of mutations within the transposon. After MmeI digestion and subsequent adapter ligation, PCR amplification and sequencing can provide information about the transposon insertion sites.
Pros:
- Can study mutational frequency of transposons
- Method can be used to deduce fitness of genes within microorganisms
- Protocol is robust, reproducible, and sensitive
Cons:
- Limited to bacterial studies
- INSeq: Goodman A. L., McNulty N. P., Zhao Y., et al. Identifying genetic determinants needed to establish a human gut symbiont in its habitat. Cell Host Microbe. 2009;6:279-289
- Tn-Seq: van Opijnen T., Bodi K. L. and Camilli A. (2009) Tn-seq: high-throughput parallel sequencing for fitness and genetic interaction studies in microorganisms. Nat Methods 6: 767-772
- Le Breton Y., Belew A. T., Valdes K. M., Islam E., Curry P., et al. (2015) Essential Genes in the Core Genome of the Human Pathogen Streptococcus pyogenes. Sci Rep 5: 9838
- Lee S. A., Gallagher L. A., Thongdee M., Staudinger B. J., Lippman S., et al. (2015) General and condition-specific essential functions of Pseudomonas aeruginosa. Proc Natl Acad Sci U S A 112: 5189-5194
- Meeske A. J., Sham L. T., Kimsey H., Koo B. M., Gross C. A., et al. (2015) MurJ and a novel lipid II flippase are required for cell wall biogenesis in Bacillus subtilis. Proc Natl Acad Sci U S A 112: 6437-6442
- Turner K. H., Wessel A. K., Palmer G. C., Murray J. L. and Whiteley M. (2015) Essential genome of Pseudomonas aeruginosa in cystic fibrosis sputum. Proc Natl Acad Sci U S A 112: 4110-4115
- Carter R., Wolf J., van Opijnen T., Muller M., Obert C., et al. (2014) Genomic analyses of pneumococci from children with sickle cell disease expose host-specific bacterial adaptations and deficits in current interventions. Cell Host Microbe 15: 587-599