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).