Abstract
Mapping-by-sequencing strategies combine next-generation sequencing (NGS) with classical linkage analysis, allowing rapid identification of the causal mutations of the phenotypes exhibited by mutants isolated in a genetic screen. Computer programs that analyze NGS data obtained from a mapping population of individuals derived from a mutant of interest to identify a causal mutation are available; however, the installation and usage of such programs requires bioinformatic skills, modifying or combining pieces of existing software, or purchasing licenses. To ease this process, we developed Easymap, an open-source program that simplifies the data analysis workflows from raw NGS reads to candidate mutations. Easymap can perform bulked segregant mapping of point mutations induced by ethyl methanesulfonate (EMS) with DNA-seq or RNA-seq datasets, as well as tagged-sequence mapping for large insertions, such as transposons or T-DNAs. The mapping analyses implemented in Easymap have been validated with experimental and simulated datasets from different plant and animal model species. Easymap was designed to be accessible to all users regardless of their bioinformatics skills by implementing a user-friendly graphical interface, a simple universal installation script, and detailed mapping reports, including informative images and complementary data for assessment of the mapping results. Easymap is available at http://genetics.edu.umh.es/resources/easymap; its Quickstart Installation Guide details the recommended procedure for installation.
Highlights
RESULTSForward genetic screens consist of random mutagenesis followed by the isolation of mutants exhibiting a phenotype of interest, and genetic analysis of these mutants to identify the mutations that cause their phenotypes
Mapping-by-sequencing strategies combine next-generation sequencing (NGS) with classical linkage analysis, allowing rapid identification of the causal mutations of the phenotypes exhibited by mutants isolated in a genetic screen
Linkage analysis of molecular markers in segregant mapping populations is the classically preferred approach to map the point mutations induced by a chemical mutagen, carried by mutants isolated in a genetic screen (Michelmore et al, 1991; Ponce et al, 1999)
Summary
Forward genetic screens consist of random mutagenesis followed by the isolation of mutants exhibiting a phenotype of interest, and genetic analysis of these mutants to identify the mutations that cause their phenotypes. We describe Easymap, an accessible graphical-interface program that analyses NGS data from mapping populations derived through a variety of experimental designs from either insertional or EMS-induced mutants. The user selects the experimental design used for mutation mapping from a variety of options for both EMS mutation mapping (backcross and outcross strategies, alternative control samples) and tagged-sequence mapping (paired-end and single-end reads). Easymap creates a comprehensive report containing highresolution images and tabular data to assist the user in interpreting the mapping results (e.g., allele frequency plots for EMS-induced mutations, RD histograms for insertional mutations, and gene plots for each putatively damaged gene; Figure 1D), a prediction of the functional effect of the candidate mutations, the flanking sequences of each mutation, and the sequences of oligonucleotide primers to genotype such mutation. We reproduced previous results for Arabidopsis thaliana (Wilson-Sánchez et al, 2014; Li W.X. et al, 2016) and Oryza sativa (Yang et al, 2013) mutants; TABLE 2 | Validation of large-insertion mapping strategies with real experimental data
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