The Essential Genome of <italic toggle="yes">Escherichia coli</italic> K-12
ABSTRACT 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 g...
| Main Authors: | , , , , , , , , |
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| Format: | Article |
| Language: | English |
| Published: |
American Society for Microbiology
2018-03-01
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| Series: | mBio |
| Subjects: | |
| Online Access: | https://journals.asm.org/doi/10.1128/mBio.02096-17 |
| _version_ | 1818840721743937536 |
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| author | Emily C. A. Goodall Ashley Robinson Iain G. Johnston Sara Jabbari Keith A. Turner Adam F. Cunningham Peter A. Lund Jeffrey A. Cole Ian R. Henderson |
| author_facet | Emily C. A. Goodall Ashley Robinson Iain G. Johnston Sara Jabbari Keith A. Turner Adam F. Cunningham Peter A. Lund Jeffrey A. Cole Ian R. Henderson |
| author_sort | Emily C. A. Goodall |
| collection | DOAJ |
| description | ABSTRACT 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. 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. 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. |
| first_indexed | 2024-12-19T04:14:41Z |
| format | Article |
| id | doaj.art-d6d581ca16844c8f95c2cee5177dcd06 |
| institution | Directory Open Access Journal |
| issn | 2150-7511 |
| language | English |
| last_indexed | 2024-12-19T04:14:41Z |
| publishDate | 2018-03-01 |
| publisher | American Society for Microbiology |
| record_format | Article |
| series | mBio |
| spelling | doaj.art-d6d581ca16844c8f95c2cee5177dcd062022-12-21T20:36:19ZengAmerican Society for MicrobiologymBio2150-75112018-03-019110.1128/mBio.02096-17The Essential Genome of <italic toggle="yes">Escherichia coli</italic> K-12Emily C. A. Goodall0Ashley Robinson1Iain G. Johnston2Sara Jabbari3Keith A. Turner4Adam F. Cunningham5Peter A. Lund6Jeffrey A. Cole7Ian R. Henderson8Institute of Microbiology and Infection, University of Birmingham, Birmingham, United KingdomInstitute of Microbiology and Infection, University of Birmingham, Birmingham, United KingdomInstitute of Microbiology and Infection, University of Birmingham, Birmingham, United KingdomInstitute of Microbiology and Infection, University of Birmingham, Birmingham, United KingdomDiscuva Ltd., Cambridge, United KingdomInstitute of Microbiology and Infection, University of Birmingham, Birmingham, United KingdomInstitute of Microbiology and Infection, University of Birmingham, Birmingham, United KingdomInstitute of Microbiology and Infection, University of Birmingham, Birmingham, United KingdomInstitute of Microbiology and Infection, University of Birmingham, Birmingham, United KingdomABSTRACT 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. 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. 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.https://journals.asm.org/doi/10.1128/mBio.02096-17Escherichia coliTraDISgenomicstn-seq |
| spellingShingle | Emily C. A. Goodall Ashley Robinson Iain G. Johnston Sara Jabbari Keith A. Turner Adam F. Cunningham Peter A. Lund Jeffrey A. Cole Ian R. Henderson The Essential Genome of <italic toggle="yes">Escherichia coli</italic> K-12 mBio Escherichia coli TraDIS genomics tn-seq |
| title | The Essential Genome of <italic toggle="yes">Escherichia coli</italic> K-12 |
| title_full | The Essential Genome of <italic toggle="yes">Escherichia coli</italic> K-12 |
| title_fullStr | The Essential Genome of <italic toggle="yes">Escherichia coli</italic> K-12 |
| title_full_unstemmed | The Essential Genome of <italic toggle="yes">Escherichia coli</italic> K-12 |
| title_short | The Essential Genome of <italic toggle="yes">Escherichia coli</italic> K-12 |
| title_sort | essential genome of italic toggle yes escherichia coli italic k 12 |
| topic | Escherichia coli TraDIS genomics tn-seq |
| url | https://journals.asm.org/doi/10.1128/mBio.02096-17 |
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