Reduced-representation sequencing methods, such as Restriction-site Associated DNA sequencing (RAD-seq), use restriction enzymes to achieve a cost-effective approach for generating genome-wide SNP data. However, a major limitation of these methods is their inability to directly assay specific loci of interest unless located near restriction sites. Here, we present ampliRAD, a novel method combining targeted (i.e., amplicon) and reduced-representation sequencing. AmpliRAD uses an initial multiplex PCR step to amplify target loci and append restriction enzyme recognition sites onto them. The PCR product is then combined with genomic DNA and used as input for a traditional RAD library preparation protocol, enabling the incorporation of virtually any target loci into a standard RAD dataset. We also introduce updates to an existing RAD protocol, including enzymatic shearing, that enhance its accessibility and efficiency. To demonstrate ampliRAD’s utility, we investigate genetic associations with adult migration timing in Dean River Chinook salmon, revealing a clear link between the GREB1L locus and migration timing that extends previous findings from southern populations to this northern river. AmpliRAD provides a powerful new tool for genomic analyses, offering the combined benefits of both reduced representation and targeted sequencing approaches.

Reduced-representation sequencing methods, such as Restriction-site Associated DNA sequencing (RAD-seq), use restriction enzymes to achieve a cost-effective approach for generating genome-wide SNP data. However, a major limitation of these methods is their inability to directly assay specific loci of interest unless located near restriction sites. Here, we present ampliRAD, a novel method combining targeted (i.e., amplicon) and reduced-representation sequencing. AmpliRAD uses an initial multiplex PCR step to amplify target loci and append restriction enzyme recognition sites onto them. The PCR product is then combined with genomic DNA and used as input for a traditional RAD library preparation protocol, enabling the incorporation of virtually any target loci into a standard RAD dataset. We also introduce updates to an existing RAD protocol, including enzymatic shearing, that enhance its accessibility and efficiency. To demonstrate ampliRAD’s utility, we investigate genetic associations with adult migration timing in Dean River Chinook salmon, revealing a clear link between the GREB1L locus and migration timing that extends previous findings from southern populations to this northern river. AmpliRAD provides a powerful new tool for genomic analyses, offering the combined benefits of both reduced representation and targeted sequencing approaches.