High-Throughput Insertion Tracking by Deep Sequencing for the Analysis of Bacterial Pathogens
Whole-genome techniques toward identification of microbial genes required for their survival and growth during infection have been useful for studies of bacterial pathogenesis. The advent of massively parallel sequencing platforms has created the opportunity to markedly accelerate such genome-scale analyses and achieve unprecedented sensitivity, resolution, and quantification. This chapter provides an overview of a genome-scale methodology that combines high-density transposon mutagenesis with a mariner transposon and deep sequencing to identify genes that are needed for survival in experimental models of pathogenesis. Application of this approach to a model pathogen, Haemophilus influenzae , has provided a comprehensive analysis of the relative role of each gene of this human respiratory pathogen in a murine pulmonary model. The method is readily adaptable to nearly any organism amenable to transposon mutagenesis.
- Site-Directed Mutagenesis for Improving Biophysical Properties of VH Domains
- Development of TLR7/8 Small RNA Antagonists
- Using T4 DNA Polymerase to Generate Clonable PCR Products
- Biallelic Gene Knockouts in Chinese Hamster Ovary Cells
- Purification of MeCP2-Containing Deacetylase from Xenopus laevis
- Assaying Chromatin Sirtuins
- Analysis of Aminoacyl- and Peptidyl-tRNAs by Gel Electrophoresis
- Identification of the Specific Binding Proteins of Bioactive Small Compound Using Affinity Resins
- Production of Infectious Poliovirus from Synthetic Viral Genomes
- Transformation of Monomorphic and Pleomorphic Trypanosoma brucei