p lead to the identification of the genes

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Pathogens
can adopt specific strategies to colonize and infect a host, or to
adapt and resist therapeutic and prophylactic interventions.
Deciphering these strategies requires determining their genetic
basis. For a given pathogen this is typically done by analyzing the
phenotype of a given gene mutant in a model of
infection/adaptation/resistance. Alternatively, random mutagenesis
can generate libraries of thousands of mutants that can be
individually tested in a phenotyping model. This can lead to the
identification of the genes required for a specific process (i.e.,
virulence, resistance). Yet, these strategies are tedious and a
limited number of infection model or conditions can be tested for a
given pathogen. Transposition-sequencing (Tn-seq, HITS, Tradis,
Inseq) has recently emerged as a way to drastically increase the
throughput of such approaches (Gawronski et al., 2009; Goodman et
al., 2009; Langridge et al., 2009; van Opijnen et al., 2009).
Transposition-sequencing (Tn-seq)
combines
transposition mutagenesis and deep-sequencing mapping and allows the
identification of mutants from an insertional library that have lost
a given function (negative selection) without the need to test
individual mutants. By monitoring a large library of single
transposon insertion mutants with high throughput sequencing, this
method can rapidly identify genomic regions that contribute to
organismal fitness under any condition that
can be assayed
in the laboratory with high resolution (Figure
1)
(van Opijnen and Camilli, 2013). For instance, a recent study defined
a “fine scale phenotype-genotype virulence map” of the human
pathogen Streptococcus
pneumoniae
by screening 17 in
vitro
and 2 in
vivo
(carriage and infection) conditions
(van Opijnen and Camilli, 2012).
Moreover, Tn-seq
can be used to query the bacterial genome with unprecedented
resolution, allowing the identification of small genes (non-coding
RNA) that may be missed in conventional screening approaches
(Barquist et al., 2013). Once a Tn-seq
library has been established for a pathogen, it can be screened in
multiple assays with near endless possibilities, from predicting
genes essential for in vitro growth to directly assaying
requirements for survival under infective conditions in vivo.
Tn-seq can be applied to determine the genes, and cellular processes,
required to resist an antibacterial treatment or to acquire new
resistance genes, to adapt to intracellular life or to compete with
other bacteria. Virtually any assay that applies a selection pressure
can be used to identify the genetic determinants involved in a
selection process.

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We here provide a protocol to conduct a Tn-seq
analysis in a Legionella pneumophila isolate. First, we
describe the procedure to generate a collection of insertion mutants
that will be suitable for a Tn-seq analysis. Several hundred
thousands mutants can be obtained with this protocol. The
transposition mutant library can then be used in any selection-based
screen. Second, we provide a detailed protocol to identify the
transposition insertion sites with the TdT method. Finally, we
provide an example of data analysis to identify the genes required
for the function tested in the selection-based screen.

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