RAD Sequencing Library Preparation

Fin tissue samples from individuals selected for RAD genotyping were used for DNA extraction using QIAGEN DNeasy 96 kits. Quantification of extract DNA was done using Invitrogen Quant-It pico green reagent and a PerkinElmer Victor V fluorimeter. Of the 456 samples chosen for inclusion in RAD library preparation, 27 had insufficient DNA concentration after extraction and quantitation. DNA extracts from the remaining 429 samples were normalized to 5 ng/μL and 500 ng of each sample was digested with Sbf1-HF restriction enzyme in NEBuffer 4 (New England Biolabs). Barcoded adapters were then ligated onto the cut ends of the restriction sites using T4 DNA ligase (New England Biolabs) and the samples were then pooled into libraries of 48 individuals each. The remaining steps of library preparation were carried out as described in Miller et al. (2012) (link) and Hecht et al. (2012) (link). The concentration of a 1:1000 dilution of each completed library was determined by quantitative polymerase chain reaction using Life Technologies PowerSYBR reagent and Kappa biosystems Illumina library DNA standards run on an Applied Biosystems 7900 instrument. Library concentration ranged from 6.5 to 71 nM after the addition of the P2 adapter and 15 cycles of PCR amplification. The concentration of each library was normalized to 5nM and sequenced on an Illumina HiSeq 2000 instrument.