Core B - Sequencing, Genotyping and Automated Mapping

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PROJECT SUMMARY/ABSTRACT Highly trained and experienced Core B laboratory personnel will be responsible for automated meiotic mapping (AMM) of mutations that alter the T1D phenotype in NOD/NckH mice (either suppressing or augmenting the normal course of T1D development). AMM is necessary for the rapid identification of single nucleotide changes responsible for phenotypes. DNA will be sent from Core C by express mail at monthly intervals, and phenotyping data will be transmitted by direct data upload on a weekly basis. Core B will first identify all coding/splicing mutations in each pedigree. Then, within each pedigree, Core B will genotype DNA from all G2 and G3 mice at pedigree-specific mutation sites to determine the zygosity of induced mutations in every mouse. Because two separate sequencing platforms are used to detect mutations (Illumina) and to genotype mutations (Ion Torrent), the latter process being performed on many individual mice, the rate of false positive mutations is essentially zero. Genotyping errors are also exceedingly rare. Genotyping failure (as distinct from error) occurs for ~2-3% of targeted loci, requiring a second attempt using capillary sequencing. The computational arm of Core B will use statistical computation, carried out on a local computer with 192 parallel processors, to identify all mutations causative of phenotype, whether they augment or suppress T1D. Software written for the parsing and display of AMM data will assure that no causative mutation goes unnoticed. Using the machine-learning program Candidate Explorer (CE), written for precisely this task, Core B will designate certain mutations for verification studies (performed in Project 2) and mechanistic studies (performed in both Project 1 and Project 2). Core B has also developed another mission-specific software tool that permits detection of epistatic interactions between mutations. This program will determine whether interactions are additive, synergistic, or antagonistic. It will also determine whether these effects are, in the case of each mutation, dominant, recessive or additive. The causative mutations identified in Core B will be interpreted in light of phenotypic studies of >230,000 mutations presently in the Mutagenetix database that were tested for immunologic effects. Just as a Coro1a mutation was shown to abolish the development of T1D (see Overall), we anticipate that mutations in many lesser-known genes may have inhibitory or augmenting effects. The Mutagenetix database will help guide our general approach to mechanism across the P01. We will explore immunologically significant mutations mechanistically with the hypothesis that they operate in cells of the immune system. The data developed in Core B will be publicly available on the Mutagenetix database through the CE program. A public version of CE is already available to display FACS data and will be adapted for the display of T1D modifier data in the course of this P01 project.