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Transposon insertion sequencing (Tn-seq) combines transposon insertional mutagenesis with massively parallel sequencing (MPS) of the transposon insertion sites to identify genes contributing to a function of interest in bacteria. The method was originally established by concurrent work in four laboratories under the acronyms HITS,[1] INSeq,[2] TraDIS,[3] and Tn-Seq.[4] Numerous variations have been subsequently developed and applied to diverse biological systems. Collectively, the methods are often termed Tn-Seq as they all involve monitoring the fitness of transposon insertion mutants via DNA sequencing approaches.[5]
Transposons are highly regulated, discrete DNA segments that can relocate within the genome. They are universal and are found in Eubacteria, Archaea, and Eukarya, including humans. Transposons have a large influence on gene expression and can be used to determine gene function. In fact, when a transposon inserts itself in a gene, the gene's function will be disrupted.[6] Because of that property, transposons have been manipulated for use in insertional mutagenesis.[7] The development of microbial genome sequencing was a major advance for the use of transposon mutagenesis.[8][9] The function affected by a transposon insertion could be linked to the disrupted gene by sequencing the genome to locate the transposon insertion site. Massively parallel sequencing allows simultaneous sequencing of transposon insertion sites in large mixtures of different mutants. Therefore, genome-wide analysis is feasible if transposons are positioned throughout the genome in a mutant collection.[5]
Transposon sequencing requires the creation of a transposon insertion library, which will contain a group of mutants that collectively have transposon insertions in all non-essential genes. The library is grown under an experimental condition of interest. Mutants with transposons inserted in genes required for growth under the test condition will diminish in frequency from the population. To identify mutants being lost, genomic sequences adjacent to the transposon ends are amplified by PCR and sequenced by MPS to determine the location and abundance of each insertion mutation. The importance of each gene for growth under the test condition is determined by comparing the abundance of each mutant before and after growth under the condition being examined. Tn-seq is useful for both the study of a single gene's fitness as well as gene interactions [10]
Signature–tagged mutagenesis (STM) is an older technique that also involves pooling transposon insertion mutants to determine the importance of the disrupted genes under selective growth conditions.[11] High-throughput versions of STM use genomic microarrays, which are less accurate and have a lower dynamic range than massively-parallel sequencing.[5] With the invention of next generation sequencing, genomic data became increasingly available. However, despite the increase in genomic data, our knowledge of gene function remains the limiting factor in our understanding of the role genes play.[12][13] Therefore, a need for a high throughput approach to study genotype–phenotype relationships like Tn-seq was necessary.
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