Transposon express, a software application to report the identity of insertions obtained by comprehensive transposon mutagenesis of sequenced genomes: analysis of the preference for in vitro tn5 transposition into gC-rich dNA

P.R. Herron, G. Hughes, G. Chandra, S. Fielding, P.J. Dyson

Research output: Contribution to journalArticle

21 Citations (Scopus)

Abstract

Comprehensive mutant libraries can be readily constructed by transposon mutagenesis. To systematically mutagenise the genome of the Gram-positive bacterium Streptomyces coelicolor A3(2), we have employed high-throughput shuttle transposon mutagenesis of a cosmid library prepared in Escherichia coli. The location of transposon insertions is determined using automated procedures for cosmid isolation and DNA sequencing. However, a major bottleneck was the subsequent analysis of DNA sequence files. To overcome this limitation, a software application, Transposon Express, was written to allow the rapid location of transposon insertions in a sequenced genome (available at http://www.swan.ac.uk/genetics/dyson/InstallTE). Transposon Express determines the identity both of a disrupted open reading frame (ORF), and the short target site duplication created by transposition. Transposon Express also reports the orientation of the transposon and can therefore predict transcriptional coupling between an upstream promoter and a promoter-less reporter gene carried by the transposon. Analysis of a large dataset of independent insertions created using a Tn5-based transposon revealed an insertional preference for GC-rich streptomycete DNA compared to E.coli vector DNA. In addition to demonstrating the value of Transposon Express as a generic tool supporting genome-wide transposon mutagenesis programs, these data provide insight into target site selection by Tn5.
LanguageEnglish
Article numberE113
JournalNucleic Acids Research
Volume32
Issue number14
DOIs
Publication statusPublished - 2004

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Mutagenesis
Cosmids
Software
Genome
DNA Sequence Analysis
Streptomyces coelicolor
Escherichia coli
DNA
Gram-Positive Bacteria
Reporter Genes
Open Reading Frames
In Vitro Techniques
Datasets

Keywords

  • transposon mutagenesis
  • genomes
  • in vitro tn5 transposition

Cite this

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title = "Transposon express, a software application to report the identity of insertions obtained by comprehensive transposon mutagenesis of sequenced genomes: analysis of the preference for in vitro tn5 transposition into gC-rich dNA",
abstract = "Comprehensive mutant libraries can be readily constructed by transposon mutagenesis. To systematically mutagenise the genome of the Gram-positive bacterium Streptomyces coelicolor A3(2), we have employed high-throughput shuttle transposon mutagenesis of a cosmid library prepared in Escherichia coli. The location of transposon insertions is determined using automated procedures for cosmid isolation and DNA sequencing. However, a major bottleneck was the subsequent analysis of DNA sequence files. To overcome this limitation, a software application, Transposon Express, was written to allow the rapid location of transposon insertions in a sequenced genome (available at http://www.swan.ac.uk/genetics/dyson/InstallTE). Transposon Express determines the identity both of a disrupted open reading frame (ORF), and the short target site duplication created by transposition. Transposon Express also reports the orientation of the transposon and can therefore predict transcriptional coupling between an upstream promoter and a promoter-less reporter gene carried by the transposon. Analysis of a large dataset of independent insertions created using a Tn5-based transposon revealed an insertional preference for GC-rich streptomycete DNA compared to E.coli vector DNA. In addition to demonstrating the value of Transposon Express as a generic tool supporting genome-wide transposon mutagenesis programs, these data provide insight into target site selection by Tn5.",
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AU - Herron, P.R.

AU - Hughes, G.

AU - Chandra, G.

AU - Fielding, S.

AU - Dyson, P.J.

PY - 2004

Y1 - 2004

N2 - Comprehensive mutant libraries can be readily constructed by transposon mutagenesis. To systematically mutagenise the genome of the Gram-positive bacterium Streptomyces coelicolor A3(2), we have employed high-throughput shuttle transposon mutagenesis of a cosmid library prepared in Escherichia coli. The location of transposon insertions is determined using automated procedures for cosmid isolation and DNA sequencing. However, a major bottleneck was the subsequent analysis of DNA sequence files. To overcome this limitation, a software application, Transposon Express, was written to allow the rapid location of transposon insertions in a sequenced genome (available at http://www.swan.ac.uk/genetics/dyson/InstallTE). Transposon Express determines the identity both of a disrupted open reading frame (ORF), and the short target site duplication created by transposition. Transposon Express also reports the orientation of the transposon and can therefore predict transcriptional coupling between an upstream promoter and a promoter-less reporter gene carried by the transposon. Analysis of a large dataset of independent insertions created using a Tn5-based transposon revealed an insertional preference for GC-rich streptomycete DNA compared to E.coli vector DNA. In addition to demonstrating the value of Transposon Express as a generic tool supporting genome-wide transposon mutagenesis programs, these data provide insight into target site selection by Tn5.

AB - Comprehensive mutant libraries can be readily constructed by transposon mutagenesis. To systematically mutagenise the genome of the Gram-positive bacterium Streptomyces coelicolor A3(2), we have employed high-throughput shuttle transposon mutagenesis of a cosmid library prepared in Escherichia coli. The location of transposon insertions is determined using automated procedures for cosmid isolation and DNA sequencing. However, a major bottleneck was the subsequent analysis of DNA sequence files. To overcome this limitation, a software application, Transposon Express, was written to allow the rapid location of transposon insertions in a sequenced genome (available at http://www.swan.ac.uk/genetics/dyson/InstallTE). Transposon Express determines the identity both of a disrupted open reading frame (ORF), and the short target site duplication created by transposition. Transposon Express also reports the orientation of the transposon and can therefore predict transcriptional coupling between an upstream promoter and a promoter-less reporter gene carried by the transposon. Analysis of a large dataset of independent insertions created using a Tn5-based transposon revealed an insertional preference for GC-rich streptomycete DNA compared to E.coli vector DNA. In addition to demonstrating the value of Transposon Express as a generic tool supporting genome-wide transposon mutagenesis programs, these data provide insight into target site selection by Tn5.

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