An ultra-dense library resource for rapid deconvolution of mutations that cause phenotypes in Escherichia coli
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,...
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Oxford University Press
2016
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| Online Access: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4797258/ |
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pubmed-47972582016-03-21 An ultra-dense library resource for rapid deconvolution of mutations that cause phenotypes in Escherichia coli Nehring, Ralf B. Gu, Franklin Lin, Hsin-Yu Gibson, Janet L. Blythe, Martin J. Wilson, Ray Bravo Núñez, María Angélica Hastings, P. J. Louis, Edward J. Frisch, Ryan L. Hu, James C. Rosenberg, Susan M. Methods Online 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. Oxford University Press 2016-03-18 2015-11-17 /pmc/articles/PMC4797258/ /pubmed/26578563 http://dx.doi.org/10.1093/nar/gkv1131 Text en © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research. http://creativecommons.org/licenses/by/4.0/ This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. |
| repository_type |
Open Access Journal |
| institution_category |
Foreign Institution |
| institution |
US National Center for Biotechnology Information |
| building |
NCBI PubMed |
| collection |
Online Access |
| language |
English |
| format |
Online |
| author |
Nehring, Ralf B. Gu, Franklin Lin, Hsin-Yu Gibson, Janet L. Blythe, Martin J. Wilson, Ray Bravo Núñez, María Angélica Hastings, P. J. Louis, Edward J. Frisch, Ryan L. Hu, James C. Rosenberg, Susan M. |
| spellingShingle |
Nehring, Ralf B. Gu, Franklin Lin, Hsin-Yu Gibson, Janet L. Blythe, Martin J. Wilson, Ray Bravo Núñez, María Angélica Hastings, P. J. Louis, Edward J. Frisch, Ryan L. Hu, James C. Rosenberg, Susan M. An ultra-dense library resource for rapid deconvolution of mutations that cause phenotypes in Escherichia coli |
| author_facet |
Nehring, Ralf B. Gu, Franklin Lin, Hsin-Yu Gibson, Janet L. Blythe, Martin J. Wilson, Ray Bravo Núñez, María Angélica Hastings, P. J. Louis, Edward J. Frisch, Ryan L. Hu, James C. Rosenberg, Susan M. |
| author_sort |
Nehring, Ralf B. |
| title |
An ultra-dense library resource for rapid deconvolution of mutations that cause phenotypes in Escherichia coli |
| title_short |
An ultra-dense library resource for rapid deconvolution of mutations that cause phenotypes in Escherichia coli |
| title_full |
An ultra-dense library resource for rapid deconvolution of mutations that cause phenotypes in Escherichia coli |
| title_fullStr |
An ultra-dense library resource for rapid deconvolution of mutations that cause phenotypes in Escherichia coli |
| title_full_unstemmed |
An ultra-dense library resource for rapid deconvolution of mutations that cause phenotypes in Escherichia coli |
| title_sort |
ultra-dense library resource for rapid deconvolution of mutations that cause phenotypes in escherichia coli |
| description |
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. |
| publisher |
Oxford University Press |
| publishDate |
2016 |
| url |
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4797258/ |
| _version_ |
1613553834880139264 |