ddRAD_Eudypteschrysolophus_schlegeli
We prepared ddRAD libraries of royal and macaroni penguins, following the protocol described in Peterson et al. (2012). Genomic DNA of each individual (500 ng) was digested using 0.5 L of EcoRI (0.1 U/ L) and 0.5 L of SphI-HF (0.1 U/ L), at 37C for three hours. Each sample was then ligated to one of 24 unique barcodes (P1). As we were working with several more samples, we formed pools of 24 individuals and to each group a second index (an illumina adapter) was used for identification, thus identification of individuals was done using two different identifiers (Peterson et al. 2012).  Then, using a Pippin Prep (Sage Science), we selected fragments of 300-400 bp. After size selection, integrity and quantification of samples was assessed using the Agilent 2100 Bioanalyzer system. Illumine adaptors (P2) were ligated for each samples and each pool was amplified using 8-10 PCR cycles (Termociclador) . Finally, we purified samples using the Thermo Scientific MagJET Separation Rack and a final quantification was performed using the fluorometer Qubit (Life technologies).