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

Tufts University, Бостон, Georgia, United States
Nature Methods (Impact Factor: 32.07). 09/2009; 6(10):767-72. DOI: 10.1038/nmeth.1377
Source: PubMed


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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    • "RW1 transcriptomics in contaminated sand SK Moreno-Forero and JR van der Meer ratio of the abundance of transposon insertions in exponentially growing RW1 populations in sand (vs the control of the transposon abundance distribution in the starter transposon library), with gene lists appearing in Supplementary Tables S14 and S15. The transposon scanning approach is based on the premise that any transposon insertion in a gene affecting growth under a specific set of growth conditions will lead to a lower abundance (or even disappearance) of that transposon mutant population in the metapopulation of all mutants (Gawronski et al., 2009; van Opijnen et al., 2009; Roggo et al., 2013). Under exclusion of essential genes and only focussing on those genes with higher expression in sand, one can conclude from GO classification that a wide range of metabolic processes is specific for growth in soil (Table 3), and a further range of gene functions is implicated in specific survival in soil upon cessation of growth (cellular homeostasis, nutrient scavenging, stress response, Table 3). "
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