nextRAD Genotyping-by-Sequencing Protocol

Genomic DNA from the 190 individuals was converted into nextRAD genotyping-by-sequencing libraries by SNPsaurus, LLC as in [41 (link)]. 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 down to fragment 3 ng of genomic DNA (the kit is optimized to fragment 50 ng). Fragmented DNA was then amplified using the Phusion Hot Start Flex DNA Polymerase (New England Biolabs, Inc., Ipswich, MA) for 25 cycles at 75°C, with one of the primers matching the adapter and extending 8 nucleotides into the genomic DNA with the selective sequence TGCAGGAG. Thus, only fragments starting with a sequence that can be hybridized by the selective sequence of the primer will be efficiently amplified. The nextRAD libraries were sequenced on a HiSeq 4000 with two lanes of 150 bp reads (University of Oregon). All sequencing reads were uploaded to the NCBI SRA database (BioProject ID: PRJNA545461).