(A) Following Illumina sequencing of barcoded fragments, sequence reads (thin lines) are aligned to a reference genome sequence (thick line). Depth of coverage varies across tags. Reads that do not align to the genome, or align in multiple locations, are discarded. (B) Sample of reads at a single RAD site. The recognition site for the enzyme Sbf1 is indicated along the reference genome sequence (top), and sequence reads typically proceed in both directions from this point, at which they overlap. At each nucleotide site, reads showing each of the four possible nucleotides can be tallied (solid blue box). (C) Nucleotide counts at each site for each individual are used in a maximum likelihood framework to assign the diploid genotype at the site. In this example, G/T heterozygote is the most likely genotype; the method provides the log-likelihood for this genotype, a maximum-likelihood estimate for the sequencing error rate ε, and a likelihood ratio test statistic comparing G/T to the second-most-likely genotype, G/G homozygote. (D) Each individual now has a diploid genotype at each nucleotide site sequenced, and single nucleotide polymorphisms (SNPs, shown in red) can be identified across populations. Note, however, that haplotype phase is still unknown across RAD tags. (E) SNPs (red ovals) are distributed across the genome (thick line), and population genetic measures (e.g. FST) are calculated for each SNP. (F) A kernel smoothing average across multiple nucleotide positions is used to produce genome-wide distributions of population genetic measures.