Abstract
ABSTRACTTransposon-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.
Highlights
Transposon-directed insertion site sequencing (TraDIS) is a highthroughput method coupling transposon mutagenesis with short-fragment DNA sequencing
In checking for uniformity of insertion density across genomic regions, we found that the density of insertions around the terminus was slightly lower than the genomic average
Comparison of the TraDIS data with data from two previous studies of E. coli K-12 under standard laboratory conditions revealed 248 genes designated essential in all three data sets (Table S2)
Summary
Transposon-directed insertion site sequencing (TraDIS) is a highthroughput method coupling transposon mutagenesis with short-fragment DNA sequencing. 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. IMPORTANCE 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. In order to resolve outstanding conflicts, we report the use of this approach to identify the essential genes of E. coli K-12 strain BW25113, a well-studied model organism for which a complete gene deletion library is available [1]
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