|BURKART-WACO, DIANA - University Of California|
|NGO, KATHIE - University Of California|
|HENRY, ISABELLE - University Of California|
|COMAI, LUCA - University Of California|
Submitted to: Book Chapter
Publication Type: Book / Chapter
Publication Acceptance Date: 3/16/2016
Publication Date: N/A
Interpretive Summary: Traditional mutagenesis is a powerful tool for generating mutations in a wide array of organisms. Populations of mutants developed by methods involving chemical or physical agents are useful for both applied (e.g., mutation breeding, genetic resource development) and basic (e.g., functional genomics) research. Determining the mutation density of individuals within a population of mutants is important for developing genetic resources that are suitable for mutation screening (i.e., reverse genetics) approaches. Using rice as a model, a protocol for generating DNA libraries of reduced complexity is described which facilitates evaluation of mutation density of a representative number of mutants by high throughput sequencing. This method provides a cost-effective way to accurately estimate the number of mutations present in a given population, which determines the suitability of the population for efficiently identifying mutations in genes of interest. This protocol also provides a way to objectively evaluate the efficacy of various mutagenic agents.
Technical Abstract: The average mutation density of a mutant population is a major consideration when developing resources for the efficient, cost-effective implementation of reverse genetics methods such as Targeting of Induced Local Lesions in Genomes (TILLING). Reliable estimates of mutation density can be achieved by the analysis of several selected loci from hundreds of individuals via mismatch cleavage of heteroduplexes or sequencing. A more rapid and less expensive alternative involves reduced representation sequencing of the genomes of a few individuals. Here we present a detailed protocol for the construction of RESCAN (restriction enzyme sequence comparative analysis) sequencing libraries using a combination of single restriction enzyme digestion and solid phase reversible immobilization-based size selection of restriction fragments. Indexing of the libraries using barcoded adapters enables cost-saving multiplexing prior to sequencing on an Illumina platform. Mutation density can be determined from the resulting sequence data with or without a reference genome.