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).