RAD-Seq of Etheostoma caeruleum: Genome-wide SNP discovery
Approx. 1 g of genomic DNA from each sample was digested at 37 C for 2 hours using high-fidelity SbfI restriction enzyme. A two-adapter process was utilized to create DNA libraries from the digested samples, with 10-15 individually barcoded samples allocated to each Illumina library pool. P1 adapters with unique barcodes were ligated to the specific SbfI overhang sequence exposed during the digestion process. Samples were then pooled, sheared using a sequence of cyclic sonication, and size-selected into approximately 300-400 base pair lengths using magnetic beads in PEG/NaCl buffer. The libraries were blunt-end repaired and dA-tailed. P2 adapters were then ligated to each pooled library to provide unique Illumina identifiers for the sequencing process. All libraries were amplified via PCR using Q5 DNA polymerase for 14 cycles, cleaned, and size-selected an additional time using a GeneRead Size Selection Kit. Genomic libraries were quantified and pooled for Illumina sequencing. DNA fragment sizes were verified using a Bioanalyzer assay and the pooled libraries were sequenced with paired-end 75 and 100 bp reads on Illumina HiSeq 4000 (75 bp paired-end) and NovaSeq 6000 (100 bp paired-end) platforms.