Methods for Genotyping-by-Sequencing.
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 simpler 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.
Department of Molecular Biology, Max Planck Institute for Developmental Biology, Spemannstrasse 37-39, 72076, Tübingen, Germany.
Beth A
BA
Rowan
Department of Molecular Biology, Max Planck Institute for Developmental Biology, Spemannstrasse 37-39, 72076, Tübingen, Germany.
Danelle K
DK
Seymour
Department of Molecular Biology, Max Planck Institute for Developmental Biology, Spemannstrasse 37-39, 72076, Tübingen, Germany.
Eunyoung
E
Chae
Department of Molecular Biology, Max Planck Institute for Developmental Biology, Spemannstrasse 37-39, 72076, Tübingen, Germany.
Derek S
DS
Lundberg
Department of Molecular Biology, Max Planck Institute for Developmental Biology, Spemannstrasse 37-39, 72076, Tübingen, Germany. weigel@tuebingen.mpg.de.
Detlef
D
Weigel
D004587:Electrophoresis, Agar Gel; D005838:Genotype; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*
GBS; Genetic mapping; Genomic DNA; RAD-seq; RESCAN; Reduced-representation; Sequencing library; Solid phase reverse immobilization