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Author Notes:

Corresponding authors: Department of Human Genetics, Emory University, 615 Michael St., Suite 301, Atlanta, GA 30322. E-mail: mzwick@emory.edu and tcaspar@emory.edu

Miao Sun and Kajari Mondal contributed equally to this work.

We are grateful to Kathryn Anderson for supplying the Nckap1khlo and Ift172wim embryos and to Jonathan Eggenschwiler for providing the Rab23opb2 embryos that we used as positive controls.

We thank members of the Caspary and Zwick labs for comments on the manuscript, Cheryl T. Strauss for editing, and the Emory-Georgia Research Alliance Genome Center (EGC), supported in part by PHS Grant UL1 RR025008 from the Clinical and Translational Science Award program, National Institutes of Health, National Center for Research Resources, for performing the Illumina sequencing runs.

Subjects:

Research Funding:

This work was supported by a Hitchings-Elion Career Award from the Burroughs Wellcome Fund (T.C.), an Emory University Research Council grant (T.C.), Ruth L. Kirschstein National Research Service Award 1F32HD060368-01A2 (V.L.H.).

Keywords:

  • Science & Technology
  • Life Sciences & Biomedicine
  • Genetics & Heredity
  • GENETICS & HEREDITY
  • N-ethyl-N-nitrosourea (ENU) mutagenesis
  • next generation sequencing
  • DNA sequencing
  • genomics
  • targeted enrichment
  • GENOMIC SELECTION
  • CAPTURE
  • ENRICHMENT
  • AMPLIFICATION
  • HYBRIDIZATION
  • MORPHOGENESIS
  • MUTAGENESIS
  • REGULATOR
  • GENETICS
  • SCREENS

Multiplex Chromosomal Exome Sequencing Accelerates Identification of ENU-Induced Mutations in the Mouse

Tools:

Journal Title:

G3

Volume:

Volume 2, Number 1

Publisher:

, Pages 143-150

Type of Work:

Article | Final Publisher PDF

Abstract:

Forward genetic screens in Mus musculus have proved powerfully informative by revealing unsuspected mechanisms governing basic biological processes. This approach uses potent chemical mutagens, such as N-ethyl-N-nitrosourea (ENU), to randomly induce mutations in mice, which are then bred and phenotypically screened to identify lines that disrupt a specific biological process of interest. Although identifying a mutation using the rich resources of mouse genetics is straightforward, it is unfortunately neither fast nor cheap. Here we show that detecting newly induced causal variants in a forward genetic screen can be accelerated dramatically using a methodology that combines multiplex chromosome-specific exome capture, next-generation sequencing, rapid mapping, sequence annotation, and variation filtering. The key innovation of our method is multiplex capture and sequence that allows the simultaneous survey of both mutant, parental, and background strains in a single experiment. By comparing variants identified in mutant offspring with those found in dbSNP, the unmutagenized background strains, and parental lines, induced causative mutations can be distinguished immediately from preexisting variation or experimental artifact. Here we demonstrate this approach to find the causative mutations induced in four novel ENU lines identified from a recent ENU screen. In all four cases, after applying our method, we found six or fewer putative mutations (and sometimes only a single one). Determining the causative variant was then easily achieved through standard segregation approaches. We have developed this process into a community resource that will speed up individual labs' ability to identify the genetic lesion in mutant mouse lines; all of our reagents and software tools are open source and available to the broader scientific community. © 2012 Sun et al.

Copyright information:

© 2012 Sun et al.

This is an Open Access work distributed under the terms of the Creative Commons Attribution 3.0 Unported License (http://creativecommons.org/licenses/by/3.0/).

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