Many molecular ecologists have used genome reduction strategies that rely upon restriction enzyme digestion of genomic DNA to sample consistent portions of the genome from individuals being studied (e.g., RADseq, GBS). Methods using restriction enzymes have many advantages, but researchers often find the existing methods expensive to initiate and difficult to implement consistently. Here, we present a low-cost and highly robust approach for the construction of dual-digest RADseq libraries. Major features of our method include: 1) minimizing the number of processing steps, 2) focusing on a single strand of the sample DNA for library construction to allow the use of a non-phosphorylated adapter on one end, 3) ligating adapters in the presence of active restriction enzymes, thereby reducing chimeras, 4) including a third restriction enzyme in the digestion reaction to cut apart adapter dimers formed by the phosphorylated adapter and thus increasing the efficiency of adapter ligation to sample DNA, 5) adapter designs that can be used interchangeably, most being compatible with multiple enzymes, 6) including variable-length internal tags within the adapters to increase the scope of sample tagging and facilitate pooling while also increasing sequence diversity when sequencing across the restriction sites, and 7) compatibility with universal dual-indexed primers which facilitate construction of combinatorial quadruple-indexed libraries that are compatible with standard Illumina sequencing reagents and libraries. We present eight adapter designs (four each for Read 1 and Read 2) that work with 72 restriction enzyme combinations, and we demonstrate the use of one set of adapters using one set of restriction enzymes across a variety of non-model organisms to discover thousands of variable loci in each species.
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