DNA-Seq of adductor muscle: hybrid abalone
A total of 200 ng genomic DNA was digested in a 30-l reaction using 8 U MspI and 8U PstI-HF (NEB) at 37  for 2 h. This was followed by an additional 20min incubation at 75 C to inactivate the restriction enzymes. Twenty microliter restriction digest was mixed with 1 l barcoded PstI-HF adapter (stock: 0.1 M), 1.5 l common MspI adapater, 4 l 10X T4-DNA ligase buffer and 200 U T4 DNA ligase (NEB) in a total volume of 40 l. Ligation was conducted at 22 C for 2 h. Followingligation, fragments smaller than 300 bp were removed by incubating the samples with 0.7 volumes of Sera-Mag SpeedBeads (GE Healthcare Life Sciences) at room temperature for 5 min. The beads were separated from the supernatant using a magnetic stand and washed three times with 200 l freshly prepared 70% ethanol. DNA was eluted from the air-dried beads with 40 l 10 mM Tris.HCl (pH 8.0). Three microliter of the resulting eluate was added to a cocktail of 16 l H2O, 5 l 5X Taq master mix (NEB), 0.5 l of a forward primer specific to the barcoded adaptor (stock: 10 M) and 0.5 l of a reverse primer with homology to the common adaptor (stock: 10 M). PCR amplification was done for each sample separately using an initial denaturation at 95 C for 30 s, 16 cycles of denaturation at 95 C for 30 s, primer annealing at 62 C for 20 s and fragment elongation at 68 C for 15 s, followed by a final fragment elongation step at 68 C for 5 min. Eight microliters of PCR product were checked on a 1.5% agarose gel. The DNA concentration of each GBS library was measured on a Qubit 2.0 using a Qubit dsDNA HS assay kit. Only GBS libraries with concentrations > 5.0 ng/l were sequenced. Thirty nanograms of each GBS library were pooled. Primers, dNTP and small DNA fragments were removed from the pooled DNA with 0.7 volumes of Sera-Mag SpeedBeads. Pooled GBS libraries (100 ng) were sequenced on an Illumina Nova platform with paired-end 323 bp reads.