1.b.3RAD genotyping of Ixodes scapularis
Before determining an optimized library preparation protocol, libraries created following normal 3RAD protocol parameters had larger DNA insert sequences than desired and less library quantity than expected. This led us to increase digestion time to shorten DNA insert size and increase ligation cycles to have a better final library yield. The resulting library preparation is relatively quick, easy, and low-cost (easy to process ≥1 set of 96 DNA samples per 2 days at ≤$5/sample; Bayona-Vasquez et al., 2019). Although the data in this work were accumulated via multiple partial HiSeqX lanes, the total number of reads is roughly equivalent to what could be obtained from a single NovaSeq S4 lane (~$5,000 U.S., in many academic core labs).
The resulting sequence quantity and quality from these libraries were typically correlated to extracted DNA quality (poorer quality DNA leading to poorer quality libraries). Only 19 samples needed to be removed from the final dataset due to poor quality libraries (missing >50% of loci). There are three I. scapularis genome assemblies, each an improvement on the previous version. Variant loci were more readily detected with each improving assembly because more sequences were mapping to the genome assembly. For this project, we used the newest reference genome (GenBank: GCA_016920785.2 ASM1692078v2), which is likely to have made large positive difference in the quality of SNPs used and our ability to identify loci of interest.