ddRAD Library preparation
DNA quality was assessed via agarose gel electrophoresis on a 1% gel, and only nondegraded DNA (as judged by a tight high molecular weight band against a lambda standard) was selected for the library preparation stage. DNA was quantified using a Qubit Broad Range dsDNA Assay (ThermoFisher Scientific) according to the manufacturer’s instructions and normalized to c. 7 ng/μl.
A total of 2 ddRAD libraries were constructed according to a modified protocol of the original (Peterson et al. , 2012) methodology. This is described in detail elsewhere (Brown et al. , 2016; Manousaki et al. , 2016). An inter and intra-library replicate sample was included during the construction of each library. Briefly, for each library individual genomic DNA (21 ng per sample) was restriction digested by Sbf I and Sph I, and then Illumina-specific sequencing adaptors (P1 & P2), each with unique combinatorial inline barcodes, were ligated to fragment ends. The samples were then pooled and size selected (400–700 bp fragments) by gel electrophoresis, PCR amplified (15 cycles) and the resultant amplicons (ddRAD library) were purified and quantified. The combinatorial inline barcodes (five or seven bases long) included in the P1 and P2 adaptors allowed each sample replicate to be demultiplexed post-sequencing. Each ddRAD library was sequenced on the Illumina MiSeq Platform (a single paired-end run; v2 chemistry, 2×150 bases).