Bulk Segregation Mapping of Mutations in Closely Related Strains of Mice

Department of Genetics, The Scripps Research Institute, La Jolla, California 92037, USA.
Genetics (Impact Factor: 5.96). 10/2010; 186(4):1139-46. DOI: 10.1534/genetics.110.121160
Source: PubMed


Phenovariance may be obscured when genetic mapping is performed using highly divergent strains, and closely similar strains are preferred if adequate marker density can be established. We sequenced the C57BL/10J mouse genome using the Applied Biosystems SOLiD platform and here describe a genome-wide panel of informative markers that permits the mapping of mutations induced on the closely related C57BL/6J background by outcrossing to C57BL/10J, and backcrossing or intercrossing. The panel consists of 127 single nucleotide polymorphisms validated by capillary sequencing: 124 spaced at ∼20-Mb intervals across the 19 autosomes, and three markers on the X chromosome. We determined the genetic relationship between four C57BL-derived substrains and used the panel to map two N-ethyl-N-nitrosourea (ENU)-induced mutations responsible for visible phenotypes in C57BL/6J mice through bulk segregation analysis. Capillary sequencing, with computation of relative chromatogram peak heights, was used to determine the proportion of alleles from each strain at each marker.

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    • "Seven homozygous B6/B10 Bishu mutant animals with the CHD/laterality defect phenotypes were used for the genome scan mapping analysis. A panel of 123 B6/B10 polymorphic DNA markers previously shown to be effective for mapping mutations was used for this analysis (Xia et al. 2010). Primers were designed to generate short DNA fragments (150–300 bp) encompassing each SNP (Supplementary Table S1) to allow the use of the Ion Torrent 200 bp sequencing chemistry. "
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    ABSTRACT: Mutation mapping in mice can be readily accomplished by genome wide segregation analysis of polymorphic DNA markers. In this study, we showed the efficacy of Ion Torrent next generation sequencing for conducting genome-wide scans to map and identify a mutation causing congenital heart disease in a mouse mutant, Bishu, recovered from a mouse mutagenesis screen. The Bishu mutant line generated in a C57BL/6J (B6) background was intercrossed with another inbred strain, C57BL/10J (B10), and the resulting B6/B10 hybrid offspring were intercrossed to generate mutants used for the mapping analysis. For each mutant sample, a panel of 123 B6/B10 polymorphic SNPs distributed throughout the mouse genome was PCR amplified, bar coded, and then pooled to generate a single library used for Ion Torrent sequencing. Sequencing carried out using the 314 chip yielded >600,000 usable reads. These were aligned and mapped using a custom bioinformatics pipeline. Each SNP was sequenced to a depth >500×, allowing accurate automated calling of the B6/B10 genotypes. This analysis mapped the mutation in Bishu to an interval on the proximal region of mouse chromosome 4. This was confirmed by parallel capillary sequencing of the 123 polymorphic SNPs. Further analysis of genes in the map interval identified a splicing mutation in Dnaic1 (c.204+1G>A), an intermediate chain dynein, as the disease causing mutation in Bishu. Overall, our experience shows Ion Torrent amplicon sequencing is high throughput and cost effective for conducting genome-wide mapping analysis and is easily scalable for other high volume genotyping analyses.
    Mammalian Genome 12/2013; 25(3-4). DOI:10.1007/s00335-013-9494-7 · 3.07 Impact Factor
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    • "Genetic mapping and whole-genome sequencing Genetic mapping was accomplished by bulk segregation analysis as previously described (Xia et al. 2010), using a panel of 127 single nucleotide polymorphisms across the genome. Once a critical region was established by genetic mapping, candidate genes within the region were sequenced. "
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    ABSTRACT: Linkage between transmembrane proteins and the spectrin-based cytoskeleton is necessary for membrane elasticity of red blood cells. Mutations of the proteins that mediate this linkage result in various types of hemolytic anemia. Here we report a novel N-ethyl-N-nitrosourea (ENU)-induced mutation of ankyrin-1, named hema6, which causes hereditary spherocytosis (HS) in mice through mild reduction of protein expression. The causal mutation was traced to a single nucleotide transition located deep into intron 13 of gene Ank1. In vitro minigene splicing assay revealed two abnormally spliced transcripts containing cryptic exons from fragments of Ank1 intron 13. The inclusion of cryptic exons introduced a premature termination codon, which leads to nonsense-mediated decay of the mutant transcripts in vivo. Hence, in homozygous mice, only wild type ankyrin-1 is expressed, albeit at 70% of the level in wild type mice. Heterozygotes display a similar HS phenotype stemming from intermediate protein expression level, indicating the haploinsufficiency of the mutation. Weakened linkage between integral transmembrane protein, band 3 and underlying cytoskeleton was observed in mutant mice owing to reduced high-affinity binding sites provided by ankyrin-1. Hema6 is the only known mouse mutant of Ank1 allelic series that expresses full-length canonical ankyrin-1 at a reduced level, a fact that makes it particularly useful to study the functional impact of ankyrin-1 quantitative deficiency.
    G3-Genes Genomes Genetics 08/2013; 3(10). DOI:10.1534/g3.113.007013 · 3.20 Impact Factor
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    • "Offspring of F1 mice were subsequently analyzed phenotypically (see below for descriptions of the phenotypic screens used) and a critical region was established by genetic linkage mapping using approximately 120 polymorphic markers across the whole genome and additional markers close to the mutation as necessary. Bulk segregation analysis (BSA) was used to map some mutations, as described [22]. Incidental mutation data were obtained by whole genome sequencing using versions 2–4 of SOLiD technology (Life Technologies, Grand Island, NY). "
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    ABSTRACT: Background: We present a compendium of N-ethyl-N-nitrosourea (ENU)-induced mouse mutations, identified in our laboratory over a period of 10 years either on the basis of phenotype or whole genome and/or whole exome sequencing, and archived in the Mutagenetix database. Our purpose is threefold: 1) to formally describe many point mutations, including those that were not previously disclosed in peer-reviewed publications; 2) to assess the characteristics of these mutations; and 3) to estimate the likelihood that a missense mutation induced by ENU will create a detectable phenotype. Findings: In the context of an ENU mutagenesis program for C57BL/6J mice, a total of 185 phenotypes were tracked to mutations in 129 genes. In addition, 402 incidental mutations were identified and predicted to affect 390 genes. As previously reported, ENU shows strand asymmetry in its induction of mutations, particularly favoring T to A rather than A to T in the sense strand of coding regions and splice junctions. Some amino acid substitutions are far more likely to be damaging than others, and some are far more likely to be observed. Indeed, from among a total of 494 non-synonymous coding mutations, ENU was observed to create only 114 of the 182 possible amino acid substitutions that single base changes can achieve. Based on differences in overt null allele frequencies observed in phenotypic vs. non-phenotypic mutation sets, we infer that ENU-induced missense mutations create detectable phenotype only about 1 in 4.7 times. While the remaining mutations may not be functionally neutral, they are, on average, beneath the limits of detection of the phenotypic assays we applied. Conclusions: Collectively, these mutations add to our understanding of the chemical specificity of ENU, the types of amino acid substitutions it creates, and its efficiency in causing phenovariance. Our data support the validity of computational algorithms for the prediction of damage caused by amino acid substitutions, and may lead to refined predictions as to whether specific amino acid changes are responsible for observed phenotypes. These data form the basis for closer in silico estimations of the number of genes mutated to a state of phenovariance by ENU within a population of G3 mice.
    BMC Research Notes 10/2012; 5(1):577. DOI:10.1186/1756-0500-5-577
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