title: Double digest RAD-Seq with NlaIII and MluCI of AoxyH15-9171
design: Briefly, two 4 base-cutting restriction enzymes NlaIII and MluCI were used to digest 200 ng DNA of each sample in separate reactions at 37 C for two hours, followed by heat inactivation of the enzyme at 65 C for 20 minutes. Five base pair unique indexed sequencing adapters were ligated to the NlaIII cut site to enable unambiguous demultiplexing of each sample after sequencing. The size selection of adapter ligated fragments was 410 base pairs (this includes primers and barcode lengths), which would result in sequenced fragments of approximately 284 bp after demultiplexing. Samples were 150 bp paired-end sequenced on an Illumina HiSeq