ddRADseq of Colossoma macropomum
25 ng of genomic DNA from each individual was double-digested (8 U/reaction) using one of two enzyme combinations, I (NlaIII and MluCI) or II (SphI and MluCI) (Table 1), and ligated to specific adapters for each enzyme (P1 and P2, 0.25 M) using T4 DNA ligase, at 23 C for 1h 30 min and 65 C for 10 min. P1 adapters had an additional 5 nucleotides that served as individual markers (barcode). Size selection of the digested fragments was performed with E-Gel SizeSelect  II (Thermo) equipment. Subsequently, PCR assays were performed to incorporate the indexes identification of each library (about 48 samples/library). Phusion enzyme conditions were used to perform the PCR assay. Reactions were purified with the AMPure XP Beads Kit and analysed by Agilent Bioanalyzer and Qubit. ddRADseq libraries were sequenced by Novogene (Sacramento, USA) on Illumina HiSeq4000 (PE 150 bp).