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.
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We thank Paulo Teixeira for sharing his modifications of the Rohland and Reich (2012) protocol for preparing the Serapure SPRI beads, and George Wang for analyzing the index sequences for the 96 whole genome adapters and indexing primers to determine optimal base balance. We also acknowledge Norman Warthmann for designing the indexing primers.
Supplementary Table 2Oligo sequences for 96 indexed P1 adapters and Universal P2 adapter If pooling fewer than 96 samples at a time, arrange the adapters in a 96-well plate according to the “Well” column and pool samples column-wise from column 1 through column 12. The 3′ phosphorothioate (PTO) modification is between the last two bases of the oligo. (XLSX 17 kb)
Supplementary Table 3Oligo sequences for PCR indexing of 96-plexes. The order column provides the optimal order for pooling 96-plexes. It is recommended to use at least four of these indices in a single lane. All oligos have a 3′phosphorothioate (PTO) modification between the last two bases of the oligo. (XLSX 39 kb)
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