RAD-Seq of pacu challenged with Aeromonas hydrophila
Each library was constructed with approximately 29 individuals identified with barcodes (14 libraries in total). Genomic DNA from each individual was digested by Sbfl enzyme. For each 11.35 l of DNA (concentration at 10 ng/l), 0.25 l of Sbfl and 1.3 l of NEBuffer 4 (New England Biolabs) were used. The enzyme digestion was performed at 37C for 1 h, followed by an inactivation period of 20 min at 80C. Barcode adapters were ligated to the end of the DNA fragments following digestion with Sbfl, using the T4 DNA ligase (New England Biolabs). The ligation was incubated at 20C for 2 h, and then at 65C for 20 min. Following library construction, their quality was determined by PCR that combined 8.5 l of water; 12.5 l Phusion High-Fidelity Master Mix; 1 l of RAD primer mix (forward and reverse) and 1 l ofdigested DNA. The amplification was carried out with 18 cycles in a thermocycler (98C for 10 s, 65C for 30 s, 72C for 30 s) and 72C for 5 min. Size selection (300 - 500 bp) was carried out using excision of the appropriate band from gel electrophoresis. Purification was then carried out by the MinElute Gel Extraction kit, following the recommendations of the manufacturer. The concentration of each library was thennormalized to 2.5 nm for sequencing.