Massively parallel sequencing of the mouse exome to accurately identify rare, induced mutations: An immediate source for thousands of new mouse models

Immunogenomics Laboratory , Australian National University , GPO Box 334, Canberra City, Australian Capital Territory, 2601 , Australia.
Open Biology (Impact Factor: 5.78). 05/2012; 2(5):120061. DOI: 10.1098/rsob.120061
Source: PubMed


Accurate identification of sparse heterozygous single-nucleotide variants (SNVs) is a critical challenge for identifying the causative mutations in mouse genetic screens, human genetic diseases and cancer. When seeking to identify causal DNA variants that occur at such low rates, they are overwhelmed by false-positive calls that arise from a range of technical and biological sources. We describe a strategy using whole-exome capture, massively parallel DNA sequencing and computational analysis, which identifies with a low false-positive rate the majority of heterozygous and homozygous SNVs arising de novo with a frequency of one nucleotide substitution per megabase in progeny of N-ethyl-N-nitrosourea (ENU)-mutated C57BL/6j mice. We found that by applying a strategy of filtering raw SNV calls against known and platform-specific variants we could call true SNVs with a false-positive rate of 19.4 per cent and an estimated false-negative rate of 21.3 per cent. These error rates are small enough to enable calling a causative mutation from both homozygous and heterozygous candidate mutation lists with little or no further experimental validation. The efficacy of this approach is demonstrated by identifying the causative mutation in the Ptprc gene in a lymphocyte-deficient strain and in 11 other strains with immune disorders or obesity, without the need for meiotic mapping. Exome sequencing of first-generation mutant mice revealed hundreds of unphenotyped protein-changing mutations, 52 per cent of which are predicted to be deleterious, which now become available for breeding and experimental analysis. We show that exome sequencing data alone are sufficient to identify induced mutations. This approach transforms genetic screens in mice, establishes a general strategy for analysing rare DNA variants and opens up a large new source for experimental models of human disease.

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    • "ENU mutations are typically SNVs and to a lesser extent, small indels. Since the emergence of NGS there has been an evolution of ENU mutation detection strategies, making ENU an efficient and attractive method to generate mouse models of human disease (Andrews et al. 2012; Potter et al. 2015). "
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    ABSTRACT: Mutagenesis-based screens in mice are a powerful discovery platform to identify novel genes or gene functions associated with disease phenotypes. An N-ethyl-N-nitrosourea (ENU) mutagenesis screen induces single nucleotide variants randomly in the mouse genome. Subsequent phenotyping of mutant and wildtype mice enables the identification of mutated pathways resulting in phenotypes associated with a particular ENU lesion. This unbiased approach to gene discovery conducts the phenotyping with no prior knowledge of the functional mutations. Before the advent of affordable next generation sequencing (NGS), ENU variant identification was a limiting step in gene characterization, akin to 'finding a needle in a haystack'. The emergence of a reliable reference genome alongside advances in NGS has propelled ENU mutation discovery from an arduous, time-consuming exercise to an effective and rapid form of mutation discovery. This has permitted large mouse facilities worldwide to use ENU for novel mutation discovery in a high-throughput manner, helping to accelerate basic science at the mechanistic level. Here, we describe three different strategies used to identify ENU variants from NGS data and some of the subsequent steps for mutation characterisation.
    Mammalian Genome 10/2015; 26(9-10). DOI:10.1007/s00335-015-9603-x · 3.07 Impact Factor
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    • "This method has allowed the successful mutation identification of a handful of mutants in flies, worms, zebrafish, and mice, but the number of complementation groups that were mapped per report is limited. It thus remains unclear how scalable this approach is or what its success rate is when one attempts to apply WGS to identify their mutant of interest (Doitsidou et al. 2010; Earley and Jones 2011; Andrews et al. 2012; Leshchiner et al. 2012; Bull et al. 2013; Henke et al. 2013). The drawback of combining meiotic mapping and WGS is that (1) recombination mapping requires several generations of back-crossing and is less straightforward when recessive lethal mutations are being mapped, and (2) in order to sequence multiple animals per genotype, animals are typically pooled and sequenced on one lane of the Illumina sequencer at a low coverage (43–53), which fails to identify many SNVs that are present in the genome. "
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    ABSTRACT: Forward genetic screens using chemical mutagens have been successful in defining the function of thousands of genes in eukaryotic model organisms. The main drawback of this strategy is the time-consuming identification of the molecular lesions causative of the phenotypes of interest. With whole-genome sequencing (WGS), it is now possible to sequence hundreds of strains, but determining which mutations are causative among thousands of polymorphisms remains challenging. We have sequenced 394 mutant strains, generated in a chemical mutagenesis screen, for essential genes on the Drosophila X chromosome and describe strategies to reduce the number of candidate mutations from an average of ∼3500 to 35 single-nucleotide variants per chromosome. By combining WGS with a rough mapping method based on large duplications, we were able to map 274 (∼70%) mutations. We show that these mutations are causative, using small 80-kb duplications that rescue lethality. Hence, our findings demonstrate that combining rough mapping with WGS dramatically expands the toolkit necessary for assigning function to genes
    Genome Research 09/2014; 24(10). DOI:10.1101/gr.174615.114 · 14.63 Impact Factor
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    • "Sequence analysis was performed by the Bioinformatics Unit of the Australian Phenomics Facility. A custom analysis pipeline was used to align the sequence reads with the reference genome (C57BL/6 NCBI m37), filter the raw single nucleotide variant (SNV) calls and generate a list of candidate SNVs as described [19]. Deep-sequencing datasets were deposited into the National Center for Biotechnology Information (NCBI) Sequence Read Archive ( with the study accession number SRP020643. "
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    ABSTRACT: CHARGE syndrome is a rare human disorder caused by mutations in the gene encoding chromodomain helicase DNA binding protein 7 (CHD7). Characteristics of CHARGE are varied and include developmental ear and hearing anomalies. Here we report a novel mouse model of CHD7 dysfunction, termed Looper. The Looper strain harbours a nonsense mutation (c.5690C>A, p.S1897X) within the Chd7 gene. Looper mice exhibit many of the clinical features of the human syndrome, consistent with previously reported CHARGE models, including growth retardation, facial asymmetry, vestibular defects, eye anomalies, hyperactivity, ossicle malformation, hearing loss and vestibular dysfunction. Looper mice display an otosclerosis-like fusion of the stapes footplate to the cochlear oval window and blepharoconjunctivitis but not coloboma. Looper mice are hyperactive and have vestibular dysfunction but do not display motor impairment.
    PLoS ONE 05/2014; 9(5):e97559. DOI:10.1371/journal.pone.0097559 · 3.23 Impact Factor
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