Computational protocol: Novel gene function revealed by mouse mutagenesis screens for models of age-related disease

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Protocol publication

[…] DNA from affected mice and littermates was prepared from ear biopsies and used for linkage mapping utilizing the Illumina GoldenGate Mouse Medium Density Linkage Panel (Gen-Probe Life Sciences Ltd, UK). DNA from G1 or affected G3 mice was prepared for either WGS using the Nucleon BACC2 Genomic DNA Extraction System (GE Healthcare Life Sciences), a library generated, and a single lane of paired-end sequencing (100 nt) undertaken employing the Illumina HiSeq platform (Oxford Genomics Centre, Wellcome Trust Centre for Human Genetics). The paired-end Illumina reads, 100 nt in length for each G1 was aligned to the reference genome (NCBIM38/mm10) using Burrows-Wheeler Aligner. Single-nucleotide variants (SNVs) in each alignment were detected using the unified GenotypeCaller in the Genome Analysis Toolkit (GATK) with mouse dbSNP version 137 as the background SNP set and default parameters. SNV sites that obtained a variant quality score <100 (this is a Phred scaled quality score, −10 × log (1−p), where p is the probability of the SNV being called incorrectly), or had a read depth <3 were removed from any further analysis. The remaining SNVs, termed high-confidence mutations, were then compared with the precompiled SNPs found in 17 inbred strains from the Mouse Genome Project and an in-house library of SNVs. The overlapping sites were removed resulting in a final list of unique ENU SNVs for each G1. These SNVs were then functionally annotated (for example, missense, intronic and so on) using NGS-SNP. All high-confidence mutations are available on MouseBook. For individual mutant lines SNVs were confirmed by Sanger sequencing of affected mice. […]

Pipeline specifications

Software tools BWA, GATK, NGS-SNP
Databases MouseBook
Application WGS analysis
Organisms Mus musculus, Homo sapiens
Chemicals Ethylnitrosourea