# AmpliRAD: A New Method Combining Amplicon and RAD Sequencing

## TL;DR

AmpliRAD is a new sequencing method that combines targeted and reduced-representation approaches to study both specific genes and genome-wide data in salmon.

## Contribution

AmpliRAD introduces a novel method that integrates amplicon and RAD sequencing to assay specific loci alongside genome-wide SNPs.

## Key findings

- AmpliRAD enables the inclusion of target loci in RAD datasets by appending restriction sites via PCR.
- The GREB1L locus is significantly associated with adult migration timing in Dean River Chinook salmon.

## Abstract

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.

We present AmpliRAD, a novel sequencing method that integrates a multiplex PCR step into traditional RAD‐seq protocols to enable the simultaneous generation of genome‐wide SNP data and targeted genotypes for specific loci. By appending restriction sites to target amplicons, the method overcomes the inability of standard RAD‐seq approaches to assay loci not located near natural restriction sites. We demonstrate the utility of this method in Dean River Chinook salmon, revealing a significant association between the GREB1L locus and adult migration timing while concurrently resolving fine‐scale population structure.