The Essential Genome of Escherichia coli K-12.
The Essential Genome of Escherichia coli K-12.
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DOI：
10.1128/mbio.02096-17
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发表时间：
2018-02-20
期刊：
影响因子：
6.4
通讯作者：
Henderson IR
中科院分区：
文献类型：
--
作者：
Goodall ECA;Robinson A;Johnston IG;Jabbari S;Turner KA;Cunningham AF;Lund PA;Cole JA;Henderson IR
转座子定向插入位点测序（TraDIS）是一种将转座子诱变与短片段DNA测序相结合的高通量方法。它通常用于识别必需基因。单基因缺失文库被认为是鉴定必需基因的金标准。目前，还没有将TraDIS方法与这些图书馆进行基准比较，因此，尚不清楚这两种方法是否具有可比性。为了解决这个问题，在大肠杆菌K-12中构建了高密度转座子文库。从该文库测序预测的必需基因与现有的必需基因数据库进行比较。为了减少必需基因的假阳性鉴定，统计数据分析包括对基因长度和基因组长度的校正。通过这种分析，新的必需基因和基因以前被错误地指定为必需的。我们表明，手动分析的TraDIS数据揭示了新的功能，不会被单独的统计分析检测。例子包括基因内的短的必需区域，方向依赖效应，以及基因组和蛋白质特征的精细分辨率识别。在转座子诱变数据集中识别这些插入概况将有助于对不太好表征的基因组进行基因组注释，并为细菌生理学和生物化学提供新的见解。确定细菌生存所必需的基因列表的动机包括确定抗菌药物开发的潜在靶点，用于生物技术开发的快速生长所需的基因，以及发现新的生化途径。为了鉴定大肠杆菌中的必需基因，我们构建了一个空前密度的转座子突变体库。对所得数据的初步自动分析显示，与文献相比，存在许多差异。我们现在报告更广泛的统计分析支持的文献检索和详细检查的高密度TraDIS测序数据的每个推定的必需基因的E。大肠杆菌模式实验室微生物。这篇论文很重要，因为它提供了对大肠杆菌必需基因的更好理解，揭示了仅依赖自动化分析的局限性，并为分析TraDIS数据提供了新的标准。
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 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 data sets will assist genome annotation of less well characterized genomes and provides new insights into bacterial physiology and biochemistry. Incentives to define lists of genes that are essential for bacterial survival include the identification of potential targets for antibacterial drug development, genes required for rapid growth for exploitation in biotechnology, and discovery of new biochemical pathways. To identify essential genes in Escherichia coli, we constructed a transposon mutant library of unprecedented density. Initial automated analysis of the resulting data revealed many discrepancies compared to the literature. We now report more extensive statistical analysis supported by both literature searches and detailed inspection of high-density TraDIS sequencing data for each putative essential gene for the E. coli model laboratory organism. This paper is important because it provides a better understanding of the essential genes of E. coli, reveals the limitations of relying on automated analysis alone, and provides a new standard for the analysis of TraDIS data.