design: We use two restriction enzymes, ApeKI (frequent cut) and BamHI HF (less frequent cut), to process genomic DNA from each individual, which is then ligated with a unique combination of 4bp ApeKI barcodes and 6 bp BamHI HF barcodes to each end of the fragments. The ligation process allows for extensive multiplexing of up to 96 individuals. A mixture of fragments from different individuals is size selected using a Pippin Prep gel (Sage Science) afterwards. Fragments in the range of 360 to 440 bp are collected and amplified using PCR, after which ddRAD library preparation is completed and is ready for quality check and sequencing. We use the Qubit 3.0 Fluorometer (Thermo Fisher Scientific) to measure the concentration of the ddRAD library and gel electrophoresis (2%  agarose gel) to check if fragments are within the intended size range. The quality of libraries is further assessed by sequencing on an Illumina MiSeq in our institute.