Format

Send to

Choose Destination
Nucleic Acids Res. 2016 Mar 18;44(5):e41. doi: 10.1093/nar/gkv1131. Epub 2015 Nov 17.

An ultra-dense library resource for rapid deconvolution of mutations that cause phenotypes in Escherichia coli.

Author information

1
Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX 77030, USA Department of Biochemistry and Molecular Biology, Baylor College of Medicine, Houston, TX 77030, USA Department of Molecular Virology and Microbiology, Baylor College of Medicine, Houston, TX 77030, USA The Dan L. Duncan Cancer Center, Baylor College of Medicine, Houston, TX 77030, USA.
2
Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX 77030, USA.
3
Deep Seq. Centre for Genetics and Genomics, Queens Medical Centre, University of Nottingham, Nottingham NG7 2UH, UK.
4
Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX 77030, USA Department of Biochemistry and Molecular Biology, Baylor College of Medicine, Houston, TX 77030, USA Department of Molecular Virology and Microbiology, Baylor College of Medicine, Houston, TX 77030, USA The Dan L. Duncan Cancer Center, Baylor College of Medicine, Houston, TX 77030, USA Undergraduate Program in Genomic Sciences, National Autonomous University of Mexico, 62210 Cuernavaca, Mexico.
5
Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX 77030, USA The Dan L. Duncan Cancer Center, Baylor College of Medicine, Houston, TX 77030, USA.
6
Department of Biochemistry and Biophysics, Texas A&M University and Texas Agrilife Research, College Station, TX 77843, USA.
7
Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX 77030, USA Department of Biochemistry and Molecular Biology, Baylor College of Medicine, Houston, TX 77030, USA Department of Molecular Virology and Microbiology, Baylor College of Medicine, Houston, TX 77030, USA The Dan L. Duncan Cancer Center, Baylor College of Medicine, Houston, TX 77030, USA smr@bcm.edu.

Abstract

With the wide availability of whole-genome sequencing (WGS), genetic mapping has become the rate-limiting step, inhibiting unbiased forward genetics in even the most tractable model organisms. We introduce a rapid deconvolution resource and method for untagged causative mutations after mutagenesis, screens, and WGS in Escherichia coli. We created Deconvoluter-ordered libraries with selectable insertions every 50 kb in the E. coli genome. The Deconvoluter method uses these for replacement of untagged mutations in the genome using a phage-P1-based gene-replacement strategy. We validate the Deconvoluter resource by deconvolution of 17 of 17 phenotype-altering mutations from a screen of N-ethyl-N-nitrosourea-induced mutants. The Deconvoluter resource permits rapid unbiased screens and gene/function identification and will enable exploration of functions of essential genes and undiscovered genes/sites/alleles not represented in existing deletion collections. This resource for unbiased forward-genetic screens with mapping-by-sequencing ('forward genomics') demonstrates a strategy that could similarly enable rapid screens in many other microbes.

PMID:
26578563
PMCID:
PMC4797258
DOI:
10.1093/nar/gkv1131
[Indexed for MEDLINE]
Free PMC Article

Supplemental Content

Full text links

Icon for Silverchair Information Systems Icon for PubMed Central
Loading ...
Support Center