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.