Gallagher Larry A
Department of Genome Sciences, University of Washington, Seattle, WA, USA.
Methods Mol Biol. 2019;1946:115-134. doi: 10.1007/978-1-4939-9118-1_12.
Transposon insertion sequencing (Tn-seq) is a powerful method for identifying genes required for virtually any growth or survival trait in bacteria. The technology employs next-generation DNA sequencing to identify and quantify the relative abundances of individual transposon mutants within complex pools of such mutants. When applied to pools of thousands to millions of random transposon mutants grown under selective pressure, the technique can rapidly identify, at genome scale, the mutants and corresponding genes negatively or positively selected. This chapter presents core protocols for Tn-seq analysis of Acinetobacter baumannii: generation of a high-saturation random transposon mutant pool, and isolation and sequencing of transposon-genome junctions from such a pool for identifying and quantifying the individual mutants. With these tools, the researcher can address diverse biological questions by carrying out selective growth of a mutant pool followed by Tn-seq analysis to identify genotype-phenotype associations.
转座子插入测序（Tn-seq）是一种强大的方法，可用于鉴定细菌中几乎任何生长或生存特性所需的基因。该技术利用下一代DNA测序来鉴定和量化此类突变体复杂文库中各个转座子突变体的相对丰度。当应用于在选择性压力下生长的数千至数百万个随机转座子突变体文库时，该技术可以在基因组规模上快速鉴定出被负向或正向选择的突变体及相应基因。本章介绍了鲍曼不动杆菌Tn-seq分析的核心方案：生成高饱和度随机转座子突变体文库，以及从该文库中分离转座子-基因组连接并进行测序，以鉴定和量化各个突变体。借助这些工具，研究人员可以通过对突变体文库进行选择性生长，然后进行Tn-seq分析以鉴定基因型-表型关联，从而解决各种生物学问题。
