title: ddRAD tags F1 hybrid individuals 1-48
design: DNA was extracted from fin tissue using the Qiagen DNeasy Blood & Tissue kit. A total of 15mg of tissue was used for each extraction, usually from fin tissue except for especially small fish where we complemented the fin with muscle tissue to make up 15mg. DNA extraction steps were performed as in the Qiagen DNeasy (C) Blood & Tissue kit protocol. After extraction the amount of DNA from each individual was quantified using spectrophotometry in the Nanodrop.     DNA was quantified using the Qubit 2.0 fluorometer (Life Technologies) and diluted with H2O to make a 40μl solution containing 1μg of genomic DNA. The DNA was then digested using two enzymes, EcoRI and MSPI, incubated at 37°C for 1 hour with ECORI and a further two hours at 37°C after adding MSPI. The double digested DNA was then cleaned using AMPure XP beads (Beckman Coulter). 48 unique barcoded P1 adapters were assigned ligated to individuals of two sets of 48. Two different P2 adapters were also ligated to differentiate the two sets of 48. Samples with unique P1 barcodes were then pooled into four sets of 12 and cleaned using AMPure XP beads. DNA was size selected for fragments between 276-324 base pairs using a Pippin Prep (Sage Science). Polymerase chain reaction (PCR) amplification using the Phusion polymerase kit (NEB) was then conducted on size selected pools. 12 cycles of  98°C for 12 seconds, 65°C for 30s, 72°C for 30 seconds were run. Six separate PCRs were done for each set of 12 individuals and these six were combined after amplification in order to avoid PCR bias. Finally, amplified DNA was cleaned and quantified. Four sets of 12 were pooled based on their P2 barcode adapter to produce two libraries of 48 individuals. Libraries were sequenced on an Illumina HiSeq 2500 at the Genomics Laboratory of the Clincal Sciences Centre at Imperial College London. Each library was run on a separate lane of a flow cell with 100 base, paired-end reads.