Double digest RAD sequencing provides flexibility in the number of homologous fragments recovered.

<p>Changing the restriction enzyme (RE) or size-selection regime modifies the fraction of genome recovered. <b>Simulation 1</b> (blue lines, shading): the expected fragment size distribution for a RE digest with NlaIII and MluCI (CATG and AATT) in the <i>Mus musculus</i> genome is shown (solid blue line). “Broad” size selection (300 bp±50 bp) is modeled by a normal sampling distribution (mean = 300 bp, SD = 25 bp). Under this sampling distribution, 4,900,000 sequence reads (dashed blue line) are expected to cover ∼119,000 regions at 7× or greater (blue area). <b>Simulation 2</b> (red lines, shading): the expected fragment size distribution for a digest with EcoRI and MspI (GAATTC and CCGG) is shown (solid red line). “Narrow” size selection (300 bp±24 bp; see text) is modeled by a normal sampling distribution (mean = 300 bp, SD = 11 bp; see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037135#pone.0037135.s003" target="_blank">Analysis S1</a> Supporting <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037135#pone-0037135-g001" target="_blank">Figure 1</a>). Under this sampling distribution, an investment of 315,000 sequence reads (dashed red line) is sufficient to recover ∼17,000 regions at 7× or greater (red area).