The essential genome of Escherichia coli K-12 identified using TraDIS. The essential genome of Escherichia coli K-12

Transposon-Directed Insertion-site Sequencing (TraDIS) is a high-throughput method coupling transposon mutagenesis with short-fragment DNA sequencing. It is commonly used to identify essential genes. Single gene deletion libraries are considered the gold standard for identifying essential genes. Currently, the TraDIS method has not been benchmarked against such libraries and therefore it remains unclear whether the two methodologies are comparable. To address this, a high density transposon library was constructed in Escherichia coli K-12. Essential genes predicted from sequencing of this library were compared to existing essential gene databases. To decrease false positive identification of essential genes, statistical data analysis included corrections for both gene length and genome length. Through this analysis new essential genes and genes previously incorrectly designated as essential were identified. We show that manual analysis of TraDIS data reveals novel features that would not have been detected by statistical analysis alone. Examples include short essential regions within genes, orientation-dependent effects and fine resolution identification of genome and protein features. Recognition of these insertion profiles in transposon mutagenesis datasets will assist genome annotation of less well characterized genomes and provides new insights into bacterial physiology and biochemistry.

转座子靶向插入位点测序（Transposon-Directed Insertion-site Sequencing, TraDIS）是一种将转座子诱变与短片段DNA测序相结合的高通量技术，常用于必需基因的鉴定。单基因敲除文库被视作鉴定必需基因的金标准。目前，TraDIS技术尚未与这类文库开展基准比对研究，因此二者的可比性尚不明确。为解决这一问题，本研究在大肠杆菌K-12中构建了高密度转座子文库，并将该文库测序所预测的必需基因与现有必需基因数据库进行了比对。为降低必需基因鉴定的假阳性率，本次统计数据分析针对基因长度与基因组长度进行了校正。通过该分析，本研究鉴定出了一批新的必需基因，以及此前被错误标注为必需基因的基因。研究表明，对TraDIS数据开展人工分析，可揭示仅靠统计分析无法捕捉到的全新特征，例如基因内部的短必需区域、取向依赖性效应，以及对基因组与蛋白质特征的高分辨率精准鉴定。在转座子诱变数据集中识别这类插入特征图谱，将有助于对特征尚不明确的基因组进行注释，并为细菌生理学与生物化学研究提供全新视角。