Sequence Capture of RADseq Libraries with Identifiable Duplicates (RADcap) Improves Genotyping and Reduces Costs

Molecular ecologists seek to genotype hundreds to thousands of loci from hundreds to thousands of individuals at minimal cost per sample. Current methods such as restriction site associated DNA sequencing (RADseq) and sequence capture are constrained by costs associated with inefficient use of sequencing data and sample preparation, respectively. I will explain RADcap, a reduced representation library preparation approach that combines the major benefits of RADseq (low cost with specific start positions) with those of sequence capture (repeatable sequencing of specific loci) to significantly increase efficiency and reduce costs relative to current approaches. The RADcap approach uses a new version of dual-digest RADseq (3RAD) to identify candidate loci for capture bait design, and subsequently uses custom sequence capture baits to consistently enrich loci from 3RAD libraries across many individuals. We combined this approach with a new method for identifying and removing PCR duplicates, which allows researchers to process RADseq data using traditional pipelines, and we assess performance on thousands of plant samples. Our results demonstrate that our RADcap method: (1) can methodologically reduce (to <5%) and computationally remove PCR duplicate reads from the resulting data; (2) produces consistently high occupancy matrices of genotypes across hundreds of individuals; (3) recovers more loci with consistent coverage at lower sequencing depth than RADseq libraries; and (4) is inexpensive, with reagent and sequencing costs totaling <$6/sample.