text: Current strategies for mutation detection in phenotype-driven screens utilising next generation sequencing. - PubMed - NCBI
  1 selected item: 26449678
  Mamm Genome. 2015 Oct;26(9-10):486-500. doi: 10.1007/s00335-015-9603-x. Epub 2015 Oct 8.
  Simon MM1, Moresco EM2, Bull KR3,4, Kumar S5, Mallon AM5, Beutler B2, Potter PK5.
  Medical Research Council Harwell (Mammalian Genetics Unit and Mary Lyon Centre), Harwell Campus, Oxfordshire, OX11 0RD, UK. m.simon@har.mrc.ac.uk.
  Center for the Genetics of Host Defense, University of Texas Southwestern Medical Center, Dallas, TX, 75390, USA.
  Nuffield Department of Medicine and Wellcome Trust Centre for Human Genetics, Oxford University, Oxford, UK.
  MRC Human Immunology Unit, Weatherall Institute of Molecular Medicine, Oxford, UK.
  Medical Research Council Harwell (Mammalian Genetics Unit and Mary Lyon Centre), Harwell Campus, Oxfordshire, OX11 0RD, UK.
  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.
  26449678
  PMC4602060
  10.1007/s00335-015-9603-x
  Overview of ENU mutation detection methods used on DNA-Seq data. Method 1 Male C57BL/6J mice mutagenized with ENU are bred to produce 50–100 third generation (G3) mice carrying mutations mostly in the heterozygous state. The G1 male founder of each pedigree is sent for whole genome sequencing. The G3 mice are put through a phenotyping screen and affected mice are genotyped with a SNP panel to identify ENU regions. Specific ENU SNPs within the candidate region are validated via Sanger Sequencing. After secondary phenotyping and inheritance testing a copy of the potential causative mutation may be generated with CRISPR/Cas9 targeting. Method 2 Two C57BL/6J mice are mutageneised with ENU, each are paired with WT C57BL/6J females to produce third generation mice carrying 4 possible haplotypes, ENU1, ENU2, WT1 and WT2. After phenotype testing 3 phenovariant G3 mice are sent for low coverage whole genome sequencing. Shared homozygous ENU variants seen in all 3 mice cluster in an IBD region, detected using the Lander-Green algorithm. Coding variants within the IBD are validated via Sanger Sequencing. Alternative alleles may be generated using CRISPR/Cas9 targeting. Method 3 Male C57BL/6J mice mutagenized with ENU are bred to produce 30–50 third generation (G3) mice carrying mutations in homozygous and heterozygous state. The G1 male founder of each pedigree is subjected to exome sequencing, and data are used to generate Ampliseq panel primers for amplification of mutated loci from G2 and G3 mouse DNA, followed by Ion PGM 200-bp sequencing. Genotyping data are uploaded to Mutagenetix prior to phenotypic screening. Quantitative phenotype data are entered into Mutagenetix and used with genotype data for mapping by Linkage Analyzer. Calculated P values for non-linkage, Manhattan plots, and scatter plots of phenotypic data for every mutant allele are displayed by Linkage Explorer. Confirmation of candidate genes depends on duplication of the mutant phenotype by a second allele, which may be generated by CRISPR/Cas9 targeting
  Current strategies for mutation detection in phenotype-driven screens utilising next generation sequencing
  Mamm Genome. 2015;26(9-10):486-500.
  Identification of ENU mutations using polymorphic markers on a mixed background. a WGS of 3 G1 samples showing heterozygous inbred SNP sites, which are shared among all samples. These sites are eliminated from the ENU mutation list; the remaining SNPs (b) are novel or ENU-induced. c Illustrates a simplistic view of randomly distributed ENU SNVs in a chromosome of a G1 mouse. The WGS of the G1 denotes the genomic location of the ENU SNVs in the candidate region of an affected G3 mouse
  Identification of IBD regions using a modified Lander–Green Algorithm, a pedigree in strain APFN1015-1017, the sequenced mice are shaded. The gene and genotype for the candidate mutation is shown for each sequenced individual. 1/1 indicates homozygous for mutation, ./. indicates insufficient coverage to call the genotype at that locus in an individual. b Plot showing IBD homozygous (red) and IBD heterozygous (blue) regions predicted by the Lander–Green-based algorithm in APFN1015-1017. c Pedigree for strain ENU22 with genotypes for the Ighm mutation. d Plot showing IBD regions for ENU22
  Presentation of mapping data by Linkage Explorer. A portion of a typical results table (top) displays P values for all three transmission models for each mutation, here sorted by phenotype. P values are linked directly to the Manhattan plot (lower left), where mousing over data points reveals the gene name and associated P value. Clicking a data point opens the scatter plot of phenotypic data graphed versus genotype (REF, homozygous for wild type allele; HET, heterozygous for mutant allele; or VAR, homozygous for mutant allele) for the mutation in question (lower right). μ mean, σ standard deviation