Determining mutation density using restriction enzyme sequence comparative analysis (RESCAN)

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 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 restriction enzyme sequence comparative analysis (RESCAN) sequencing libraries using a combination of single restriction enzyme digestion and solid phase reversible immobilizationbased 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.