TY - JOUR
T1 - Real-time resolution of point mutations that cause phenovariance in mice
AU - Wang, Tao
AU - Zhan, Xiaowei
AU - Bua, Chun Hui
AU - Lyona, Stephen
AU - Pratta, David
AU - Hildebrand, Sara
AU - Choi, Jin Huk
AU - Zhang, Zhao
AU - Zeng, Ming
AU - Wang, Kuan Wen
AU - Turer, Emre E
AU - Chen, Zhe
AU - Zhang Ph.D., Duan-Wu
AU - Yue, Tao
AU - Wang, Ying
AU - Shi, He-Xin
AU - Wang, Jianhui
AU - Sun, Lei
AU - SoRelle, Jeff
AU - McAlpine, William
AU - Hutchins, Noelle
AU - Zhan, Xiaoming
AU - Fin, Maggy
AU - Gobert, Rochelle
AU - Quan, Jiexia
AU - Kreutzer, McKensie
AU - Arnett, Stephanie
AU - Hawkins, Kimberly
AU - Leach, Ashley
AU - Tate, Christopher
AU - Daniel, Chad
AU - Reyna, Carlos
AU - Prince, Lauren
AU - Davis, Sheila
AU - Purrington, Joel
AU - Bearden, Rick
AU - Weatherly, Jennifer
AU - White, Danielle
AU - Russell, Jamie L
AU - Sun, Qihua
AU - Tang, Miao
AU - Li, Xiaohong
AU - Scott, Lindsay
AU - Moresco, Eva Marie Y
AU - McInerney, Gerald M.
AU - Karlsson Hedestam, Gunilla B.
AU - Xie, Yang
AU - Beutler, Bruce A
PY - 2015/2/3
Y1 - 2015/2/3
N2 - With the wide availability of massively parallel sequencing technologies, genetic mapping has become the rate limiting step in mammalian forward genetics. Here we introduce a method for real-time identification of N-ethyl-N-nitrosourea-induced mutations that cause phenotypes in mice. All mutations are identified by whole exome G1 progenitor sequencing and their zygosity is established in G2/G3 mice before phenotypic assessment. Quantitative and qualitative traits, including lethal effects, in single or multiple combined pedigrees are then analyzed with Linkage Analyzer, a software program that detects significant linkage between individual mutations and aberrant phenotypic scores and presents processed data as Manhattan plots. As multiple alleles of genes are acquired through mutagenesis, pooled "superpedigrees" are created to analyze the effects. Our method is distinguished from conventional forward genetic methods because it permits (1) unbiased declaration of mappable phenotypes, including those that are incompletely penetrant (2), automated identification of causative mutations concurrent with phenotypic screening, without the need to outcross mutant mice to another strain and backcross them, and (3) exclusion of genes not involved in phenotypes of interest. We validated our approach and Linkage Analyzer for the identification of 47 mutations in 45 previously known genes causative for adaptive immune phenotypes; our analysis also implicated 474 genes not previously associated with immune function. The method described here permits forward genetic analysis in mice, limited only by the rates of mutant production and screening.N-ethyl-N-nitrosourea, genetic mapping, forward genetics, mutagenesis, massively parallel sequencing.
AB - With the wide availability of massively parallel sequencing technologies, genetic mapping has become the rate limiting step in mammalian forward genetics. Here we introduce a method for real-time identification of N-ethyl-N-nitrosourea-induced mutations that cause phenotypes in mice. All mutations are identified by whole exome G1 progenitor sequencing and their zygosity is established in G2/G3 mice before phenotypic assessment. Quantitative and qualitative traits, including lethal effects, in single or multiple combined pedigrees are then analyzed with Linkage Analyzer, a software program that detects significant linkage between individual mutations and aberrant phenotypic scores and presents processed data as Manhattan plots. As multiple alleles of genes are acquired through mutagenesis, pooled "superpedigrees" are created to analyze the effects. Our method is distinguished from conventional forward genetic methods because it permits (1) unbiased declaration of mappable phenotypes, including those that are incompletely penetrant (2), automated identification of causative mutations concurrent with phenotypic screening, without the need to outcross mutant mice to another strain and backcross them, and (3) exclusion of genes not involved in phenotypes of interest. We validated our approach and Linkage Analyzer for the identification of 47 mutations in 45 previously known genes causative for adaptive immune phenotypes; our analysis also implicated 474 genes not previously associated with immune function. The method described here permits forward genetic analysis in mice, limited only by the rates of mutant production and screening.N-ethyl-N-nitrosourea, genetic mapping, forward genetics, mutagenesis, massively parallel sequencing.
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U2 - 10.1073/pnas.1423216112
DO - 10.1073/pnas.1423216112
M3 - Article
C2 - 25605905
AN - SCOPUS:84922219314
SN - 0027-8424
VL - 112
SP - E440-E449
JO - Proceedings of the National Academy of Sciences of the United States of America
JF - Proceedings of the National Academy of Sciences of the United States of America
IS - 5
ER -