2.2 DNA extraction, library preparation, and
sequencing
Total genomic DNA was extracted from mesothorax muscle tissues of each
beetle for library preparation and sequencing using the TruSeq DNA
Sample Preparation kit (Illumina, USA)according to the manufacturer’s
instructions. Sequencing libraries were generated using the Truseq Nano
DNA HT Sample Preparation kit (Illumina, USA) following the
manufacturer’s recommendations, and index codes were added to label
sequences from each sample. For each individual, the DNA sample was
sheared into fragments of 350 bp fragments by using a Covaris S2
(Covaris). The DNA fragments were end-polished, A-tailed, and ligated
with a full-length adapter for Illumina sequencing with further PCR
amplification. The PCR products were purified (AMPure XP system), and
the libraries were analyzed for size distribution using an Agilent 2100
Bioanalyzer and quantified using real-time PCR. The DNA fragments were
then processed and sequenced using the Illumina HiSeq PE150 platform.
Quality control was achieved by removing low costs and low-quality
paired reads (reads with ≥10% unidentified nucleotides;
>10 nt aligned to the adaptor, allowing ≤10% mismatches;
>50% of bases having phred quality <5; and
putative PCR duplicates generated in the library construction process),
which mainly resulted from base-calling duplicates and adaptor
contamination.