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van Opijnen T, Bodi KL, Camilli A.. Tn-seq: high-throughput parallel sequencing for fitness and genetic interaction studies in microorganisms. Nat Methods 6: 767-772

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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    • "59 Epistasis has also been studied in vivo. For example, in yeast, the effects of specific mutations on 60 fitness can be rapidly analyzed in the background of thousands of individual gene knockouts 61 using the epistatic mini-array profile (E-MAP) approach (Schuldiner et al. 2005), or synthetic 62 genetic analysis (SGA, Tong et al. 2001; van Opijnen et al. 2009). While epistasis mapping by 63 these approaches has been extremely useful for detecting physiological connections between 64 gene products, it is not well suited to investigate intragenic epistasis, or to comprehensively 65 screen point mutants. "
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    • "One of the potential perils of using next generation sequencing, with its current reliance on short read sequences is that transposition events may be missed. Therefore, employing methods that specifically identify novel insertion sites of retrotransposon (Gabriel et al. 2006; Mularoni et al. 2012; van Opijnen et al. 2009) remains an important aspect of analyzing genomes of evolved lineages. "
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    • "Essential protein-coding sequences have been reported for several bacterial species using relatively low-throughput transposon mutagenesis (Hutchison et al, 1999; Jacobs et al, 2003; Glass et al, 2006) and in-frame deletion libraries (Kobayashi et al, 2003; Baba et al, 2006). Two recent studies used high-throughput transposon mutagenesis for fitness and genetic interaction analysis (Langridge et al, 2009; van Opijnen et al, 2009). Here, we have reliably identified all essential coding and non-coding chromosomal elements, using a hyper-saturated transposon mutagenesis strategy that is scalable and can be extended to obtain rapid and highly accurate identification of the entire essential genome of any bacterial species at a resolution of a few base pairs. "
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