Next-RAD of Gila robusta
All DNA extractions from this study were coded such that samples could be run blindly without knowledge of the species ID or site of origin through nextRAD (Nextera-tagmented, reductively-amplified DNA) genotyping-by-sequencing to collect SNP data. This nextRAD approach uses selective PCR primers to amplify genomic loci consistently between samples. Genomic DNA was first fragmented with Nextera reagent (Illumina, Inc, San Diego, CA, USA), which also ligates short adapter sequences to the ends of the fragments. The Nextera reaction was scaled for fragmenting 15 ng of genomic DNA, although 60 ng of genomic DNA was used for input to compensate for degraded DNA in the samples and to increase fragment sizes. Fragmented DNA was then amplified for 27 cycles at 74 C, with one of the primers matching the adapter and extending 10 nucleotides into the genomic DNA with the selective sequence GTGTAGAGCC. Thus, only fragments starting with a sequence that can be hybridized by the selective sequence of the primer will be efficiently amplified. The resulting fragments are fixed at the selective end and have random lengths depending on the initial Nextera fragmentation. Because of this, amplified DNA from a particular locus is present at many different sizes and careful size selection of the library is not needed prior to sequencing. These nextRAD libraries were sequenced on a HiSeq 4000 with four lanes of 150 bp reads at the University of Oregon (Eugene, OR, USA).