Targeted deep sequencing
Target sites were first amplified to a size of ~500 bp
from extracted genomic DNA using KAPA HiFi HotStart DNA polymerase
(Roche, Cat. no. #KK2502) according to the manufacturer’s protocols.
Then, amplicons were amplified again to a size of ~230
bp, after which the amplicons were amplified using TruSeq HT dual
index-containing primers to add adaptor and index sequences for Illumina
sequencing platforms to each sample [20]. Primers used in this study
are listed in Supplementary Table 3. Pooled PCR amplicons were purified
using a PCR purification kit (MGmed) and sequenced on a MiniSeq
(Illumina) with paired-end sequencing systems (2x150 bp). Cas-Analyzer
(http://www.rgenome.net/cas-analyzer/#!) was used to quantify the indel
frequencies from deep sequencing data [20].