期刊
NUCLEIC ACIDS RESEARCH
卷 44, 期 5, 页码 -出版社
OXFORD UNIV PRESS
DOI: 10.1093/nar/gkv1131
关键词
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资金
- National Institutes of Health (NIH) [DP1-CA174424]
- National Institute of General Medical Sciences [R01-GM53158, F32-GM095267, U24GM088849]
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.
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