A GENOME-WIDE CATALOGUE OF SINGLE NUCLEOTIDE POLYMORPHISMS IN WHITE-BEAKED AND ATLANTIC WHITE-SIDED DOLPHINS: Sample GER_18088
"RADSeq libraries were built following a customized protocol based on Baird et al. (2008). In brief, 1 microg of genomic DNA was digested at 37C overnight for at least 14 hours in a 50 microl reaction with 40-45 units of the restriction enzyme SbfI-HF (NEB). For each sample, the digested product was ligated to a P1 6bp-barcoded adapter (Table S2) through a 60 microl reaction containing 1,000 units of T4 ligase (NEB) and 1mM rATP. Barcodes were selected to differ by a minimum of 2 nucleotides to minimize sample miss-classification due to sequencing errors. After P1 adaptor-ligation, libraries combining the products of four samples (2 microg total DNA) were built and sheared in a bioruptor (Diagenode) using 4 cycles of 30 seconds on and 90 seconds off, with power settings on high. In one occasion, the library contained products of two samples only (GER_16411 and GER_3614). Whenever possible, samples with DNA extracts showing similar concentration were merged in the same library to avoid over-representing a particular sample. Fragmented products were size-selected to target 300-600bp-long templates using Agencourt AMPure XP magnetic beads. Efficiency of the size-selection procedure was assessed using 2% agarose gels. Selected fragments were blunt ended (Quick blunting kit, NEB) and A-tailed (Klenow fragments, 3' to 5' exo, NEB) following manufacturer's instructions. A-tailed fragments were ligated to a P2 adapter (Table S2) through a 50 microl reaction containing 1000 units of T4 ligase (NEB) and 1mM rATP.PCR reactions of 25 microl total volume were carried out and consisted in 1X AmpliTag Gold PCR buffer, 4mM MgCl2, 1 microg BSA, 2.5 units AmpliTaq Gold (Life Technologies), 1mM dNTP, 0.5 microM of forward and reverse Illumina TruSeq primers (Table S2) and 14.25 microl library template. After an initial denaturation at 92C for 10 minutes, we performed 26-28 amplification cycles of 92C for 30 seconds, 60C for 30 seconds, 72C for 40 seconds and a final extension of 72C for 7 minutes. Libraries were pooled (8-10 libraries per lane) and sequenced as Single Reads on an Illumina HiSeq2000 platform at the Danish National High-throughput DNA Sequencing Centre."