Rackerby Bryna, Lee Sang In, Moppert Ian, Ricke Steven C, Jeong KwangCheol C, Park Si Hong
Department of Microbiology, Oregon State University, Corvallis, OR, USA.
Department of Food Science and Technology, Oregon State University, Corvallis, OR, USA.
Methods Mol Biol. 2019;2016:17-27. doi: 10.1007/978-1-4939-9570-7_2.
Transposons are elements widely dispersed among organisms which are able to move and replicate fragments of genomes. The extensive variability in transposons present in most organisms requires extensive identification and interpretation of the resulting transposon mutants after transposon mutagenesis. However, much of this is reliant on utilizing randomness characteristics of transposon to identify essential genes for the organism of interest. Integration of the transposon mutant approach with commercialized next-generation sequencing (NGS) technology has helped to advance transposon identification by sequencing millions of reads generated from a single run on an NGS platform. Transposon sequencing is defined as a gene sequencing methodology that allows for the identification of nonessential genes and the determination of gene function using a random transposon insertional mutagenesis followed by massively parallel sequencing. The detailed protocol will be outlined in this chapter. The genomic DNA integrated with the transposons is sequenced using an NGS platform in order to determine the location of each mutation.
转座子是广泛分布于生物体中的元件，能够移动和复制基因组片段。大多数生物体中存在的转座子具有广泛的变异性，这就需要在转座子诱变后对产生的转座子突变体进行广泛的鉴定和解读。然而，这很大程度上依赖于利用转座子的随机性特征来鉴定目标生物体的必需基因。将转座子突变体方法与商业化的下一代测序（NGS）技术相结合，通过对在NGS平台上单次运行产生的数百万条读数进行测序，有助于推进转座子鉴定。转座子测序被定义为一种基因测序方法，它允许通过随机转座子插入诱变，随后进行大规模平行测序来鉴定非必需基因并确定基因功能。本章将概述详细的实验方案。对整合有转座子的基因组DNA使用NGS平台进行测序，以确定每个突变的位置。
