221 Stefan J. White and Stuart Cantsilieris (eds.), Genotyping: Methods and Protocols, Methods in Molecular Biology, vol. 1492, DOI 10.1007/978-1-4939-6442-0_16, © Springer Science+Business Media New York 2017 Chapter 16 Methods for Genotyping-by-Sequencing Beth A. Rowan, Danelle K. Seymour, Eunyoung Chae, Derek S. Lundberg, and Detlef Weigel Abstract A major goal for biologists is to understand the connection between genes and phenotypic traits, and genetic mapping in experimental populations remains a powerful approach for discovering the causal genes underlying phenotypes. For genetic mapping, the process of genotyping was previously a major rate- limiting step. Modern sequencing technology has greatly improved the resolution and speed of genetic mapping by reducing the time, labor, and cost per genotyping marker. In addition, the ability to perform genotyping-by-sequencing (GBS) has facilitated large-scale population genetic analyses by providing a sim- pler way to survey segregating genetic variation in natural populations. Here we present two protocols for GBS, using the Illumina platform, that can be applied to a wide range of genotyping projects in different species. The first protocol is for genotyping a subset of marker positions genome-wide using restriction digestion, and the second is for preparing inexpensive paired-end whole-genome libraries. We discuss the suitability of each approach for different genotyping applications and provide notes for adapting these protocols for use with a liquid-handling robot. Key words Genomic DNA, Reduced-representation, Genetic mapping, RAD-seq, GBS, RESCAN, Sequencing library, Solid phase reverse immobilization 1 Introduction Genotyping is an essential component of many different avenues of research in biology. For example, whether the investigator is con- cerned with understanding the genetic basis of phenotypic traits or the processes of molecular evolution, the ease of obtaining geno- types is one factor that affects the number of individuals that can be feasibly included in an experiment. The cost of high-throughput short-read sequence data has dropped precipitously over the past decade, owing to continued improvements to modern sequencing platforms. The Illumina Electronic supplementary material: The online version of this chapter (doi:10.1007/978-1-4939-6442-0_16) contains supplementary material, which is available to authorized users.