RADseq Library Preparation and Sequencing
Quadruple indexed 3RAD libraries were created for every individual sample following a protocol modified from Adapterama III (Bayona-Vasquez et al., 2019) using Xbal, EcoRI-HF, and NheI-HF enzyme combination (New England Biolabs, Inc., MA, USA). In this protocol, XbaI and EcoRI-HF provide the cut sites for adapter ligation to the target DNA, whereas NheI-HF is used to cut adapter-dimers and increase efficiency of library preparation which is particularly important when only limited amounts of target DNA are available. Modifications were as follows: target DNA concentration was at a lower starting concentration than the recommended 20ng/µL, digestion time was increased from one hour to two hours, ligation cycles were increased to seven total cycles, and libraries were not cleaned between ligation and PCR steps as this does not impact library quality while saving time and reagents.
Libraries were completed by amplifying the uncleaned ligation product with 13 cycles of PCR using iTru5 and iTru7 primers (primer sequences from Glenn et al., 2019). We then visualized the libraries on a 1.5% agarose gel to ensure an appropriate size-distribution of library molecules and to screen for irregularities, which we did not observe. Individual barcoded libraries were pooled and cleaned using Speed-Beads at a 1:1.25 ratio (Pool:Speed-Beads). The cleaned pool was further size selected on a Pippin Prep (Sage Science, MA, USA) using a 1.5% dye-free Marker K agarose gel cassette (CDF1510) set to capture fragments of 550 bp +/-10%. P5 and P7 Illumina primers were used in a final PCR followed by another cleaning with Speed-Beads (see Glenn et al. 2019 for details). These pools were sequenced on an Illumina HiSeqX to obtain PE150 reads (NovoGene Co., Sacramento, CA).