Tn-seq; high-throughput parallel sequencing for fitness and genetic interaction studies in microorganisms

Howard Hughes Medical Institute, and Department of Molecular Biology and Microbiology, Tufts University School of Medicine, Boston, Massachusetts, USA.
Nature Methods (Impact Factor: 25.95). 09/2009; 6(10):767-72. DOI: 10.1038/nmeth.1377
Source: PubMed

ABSTRACT Biological pathways are structured in complex networks of interacting genes. Solving the architecture of such networks may provide valuable information, such as how microorganisms cause disease. Here we present a method (Tn-seq) for accurately determining quantitative genetic interactions on a genome-wide scale in microorganisms. Tn-seq is based on the assembly of a saturated Mariner transposon insertion library. After library selection, changes in frequency of each insertion mutant are determined by sequencing the flanking regions en masse. These changes are used to calculate each mutant's fitness. Using this approach, we determined fitness for each gene of Streptococcus pneumoniae, a causative agent of pneumonia and meningitis. A genome-wide screen for genetic interactions of five query genes identified both alleviating and aggravating interactions that could be divided into seven distinct categories. Owing to the wide activity of the Mariner transposon, Tn-seq has the potential to contribute to the exploration of complex pathways across many different species.

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