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

Andrews TD, Whittle B, Field MA, Balakishnan B, Zhang Y, Shao Y, Cho V, Kirk M, Singh M, Xia Y, Hager J, Winslade S, Sjollema G, Beutler B, Enders A, Goodnow CC - Open Biol (2012)

Bottom Line: 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.We show that exome sequencing data alone are sufficient to identify induced mutations.

View Article: PubMed Central - PubMed

Affiliation: Immunogenomics Laboratory, Australian National University, GPO Box 334, Canberra City, Australian Capital Territory, 2601 , Australia. dan.andrews@anu.edu.au

ABSTRACT
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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Summary of the structure of ENU-mutated mouse pedigrees. Each pedigree is initiated by two unrelated G1 founders. Each of these founders inherits a random set of de novo point mutations (coloured circles) on the paternal chromosomes, induced by ENU treatment of their male parent. These G1 founders will carry on average one to two DNA variants per Mb and 90 exonic ENU-induced mutations. Second-generation (G2) progeny of these mice inherit a theoretical 45 ENU-induced exonic mutations, all of which are carried in the heterozygous state. Two productive sibling–sibling matings of the G2 mice result in third-generation (G3) progeny that carry approximately 94% of the founding ENU-induced, protein-coding mutations, of which on average five are homozygous in any given mouse.
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RSOB120061F1: Summary of the structure of ENU-mutated mouse pedigrees. Each pedigree is initiated by two unrelated G1 founders. Each of these founders inherits a random set of de novo point mutations (coloured circles) on the paternal chromosomes, induced by ENU treatment of their male parent. These G1 founders will carry on average one to two DNA variants per Mb and 90 exonic ENU-induced mutations. Second-generation (G2) progeny of these mice inherit a theoretical 45 ENU-induced exonic mutations, all of which are carried in the heterozygous state. Two productive sibling–sibling matings of the G2 mice result in third-generation (G3) progeny that carry approximately 94% of the founding ENU-induced, protein-coding mutations, of which on average five are homozygous in any given mouse.

Mentions: Many parallel mouse pedigrees, each segregating a different set of random, de novo mutations induced in the C57BL/6j genome by ENU were established using the breeding strategy shown in figure 1. Each pedigree was founded by two unrelated G1 mice conceived from male C57BL/6j mice that had been treated with three doses of ENU administered at 90 mg kg−1 to induce random point mutations in spermatogonial stem cells [2,10,11]. Based on published mutation rates [12–14], we estimated that each of these G1 animals would carry approximately one de novo SNV per Mb of the paternal genome, of which around 45 would result in a non-synonymous exonic mutation. Intercrossing of the G1 animals transmitted half of these mutations in heterozygous state to each of their second-generation (G2) offspring. Intercrossing the G2 animals subsequently transmitted approximately 94 per cent of the mutations to offspring, a subset of which was inherited in homozygous state in third-generation (G3) animals (figure 1).Figure 1.


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

Andrews TD, Whittle B, Field MA, Balakishnan B, Zhang Y, Shao Y, Cho V, Kirk M, Singh M, Xia Y, Hager J, Winslade S, Sjollema G, Beutler B, Enders A, Goodnow CC - Open Biol (2012)

Summary of the structure of ENU-mutated mouse pedigrees. Each pedigree is initiated by two unrelated G1 founders. Each of these founders inherits a random set of de novo point mutations (coloured circles) on the paternal chromosomes, induced by ENU treatment of their male parent. These G1 founders will carry on average one to two DNA variants per Mb and 90 exonic ENU-induced mutations. Second-generation (G2) progeny of these mice inherit a theoretical 45 ENU-induced exonic mutations, all of which are carried in the heterozygous state. Two productive sibling–sibling matings of the G2 mice result in third-generation (G3) progeny that carry approximately 94% of the founding ENU-induced, protein-coding mutations, of which on average five are homozygous in any given mouse.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3376740&req=5

RSOB120061F1: Summary of the structure of ENU-mutated mouse pedigrees. Each pedigree is initiated by two unrelated G1 founders. Each of these founders inherits a random set of de novo point mutations (coloured circles) on the paternal chromosomes, induced by ENU treatment of their male parent. These G1 founders will carry on average one to two DNA variants per Mb and 90 exonic ENU-induced mutations. Second-generation (G2) progeny of these mice inherit a theoretical 45 ENU-induced exonic mutations, all of which are carried in the heterozygous state. Two productive sibling–sibling matings of the G2 mice result in third-generation (G3) progeny that carry approximately 94% of the founding ENU-induced, protein-coding mutations, of which on average five are homozygous in any given mouse.
Mentions: Many parallel mouse pedigrees, each segregating a different set of random, de novo mutations induced in the C57BL/6j genome by ENU were established using the breeding strategy shown in figure 1. Each pedigree was founded by two unrelated G1 mice conceived from male C57BL/6j mice that had been treated with three doses of ENU administered at 90 mg kg−1 to induce random point mutations in spermatogonial stem cells [2,10,11]. Based on published mutation rates [12–14], we estimated that each of these G1 animals would carry approximately one de novo SNV per Mb of the paternal genome, of which around 45 would result in a non-synonymous exonic mutation. Intercrossing of the G1 animals transmitted half of these mutations in heterozygous state to each of their second-generation (G2) offspring. Intercrossing the G2 animals subsequently transmitted approximately 94 per cent of the mutations to offspring, a subset of which was inherited in homozygous state in third-generation (G3) animals (figure 1).Figure 1.

Bottom Line: 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.We show that exome sequencing data alone are sufficient to identify induced mutations.

View Article: PubMed Central - PubMed

Affiliation: Immunogenomics Laboratory, Australian National University, GPO Box 334, Canberra City, Australian Capital Territory, 2601 , Australia. dan.andrews@anu.edu.au

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus