Data from: Adapterama III: quadruple-indexed, double/triple-enzyme RADseq libraries (2RAD/3RAD)

Molecular ecologists frequently use genome reduction strategies that rely
upon restriction enzyme digestion of genomic DNA to sample consistent
portions of the genome from many individuals (e.g., RADseq, GBS). However,
researchers often find the existing methods expensive to initiate and/or
difficult to implement consistently, especially due to the inability to
highly-multiplex samples to fill entire sequencing lanes. Here, we
introduce a low-cost and highly robust approach for the construction of
dual-digest RADseq libraries that relies on adapters and primers designed
in Adapterama I. Major features of our method include: 1) minimizing the
number of processing steps; 2) focusing on a single strand of sample DNA
for library construction, allowing 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 an optional third
restriction enzyme to cut apart adapter-dimers formed by the
phosphorylated adapter, thus increasing the efficiency of adapter ligation
to sample DNA, which is particularly effective when only low
quantity/quality DNA samples are available; 5) interchangeable adapter
designs; 6) incorporating variable-length internal indexes within the
adapters to increase the scope of sample indexing, facilitate pooling, and
increase sequence diversity; 7) maintaining compatibility with universal
dual-indexed primers and thus, Illumina sequencing reagents and libraries;
and, 8) easy modification for the identification of PCR duplicates. We
present eight adapter designs that work with 72 restriction enzyme
combinations. We demonstrate the efficiency of our approach by comparing
it with existing methods, and we validate its utility through the
discovery of many variable loci in a variety of non-model organisms. Our
2RAD/3RAD method is easy to perform, has low startup costs, produces
libraries that can be highly-multiplexed and pooled with other Illumina
libraries, and 3RAD has increased utility with low-concentration input
DNA.