Mapping-by-Sequencing of Point and Insertional Mutations with Easymap.

Random mutagenesis followed by screening for phenotypes of interest is a widely used strategy for genetic dissection of biological pathways; however, identifying the causal gene traditionally required time-consuming mapping approaches based on iterative linkage analysis. Mapping-by-sequencing accelerates this process, efficiently linking the phenotype of a mutant to a narrow candidate genomic region, using next-generation sequencing (NGS) data from a mapping population segregating for the mutant phenotype. To enable researchers at any bioinformatics skill level to conduct mapping-by-sequencing, we developed the Easymap mapping software. In this protocol we break down the steps involved in mapping-by-sequencing. First, we describe different ways of obtaining a mapping population and the steps used to generate NGS data. Next, we show how to analyze the NGS data using Easymap and how to obtain a list of candidate mutations, along with comprehensive information for assessing the potential causality of each candidate. Thus, this protocol enables the user to conduct mapping-by-sequencing using Easymap, facilitating the identification of causal loci for a mutant phenotype of interest.