Fig. 1.

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