title: Pacbio CCS of Epinephelus awoara: muscle
design: SMRTbell target size libraries were created for sequencing following PacBio's standard protocol (Pacific Biosciences, CA, USA), using either 10 kb or 20 kb preparation solutions. The key steps for library preparation include: (1) shearing of genomic DNA (gDNA), (2) repairing DNA damage, (3) ligating DNA fragments with hairpin adapters from the SMRTbell Express Template Prep Kit 2.0 (Pacific Biosciences), (4) selecting fragments of the desired size, and (5) binding to a polymerase. In brief, 2 micrograms (2 g) of DNA per sample were used for DNA library preparations. Genomic DNA samples were sheared to achieve the expected fragment size using g-TUBEs (Covaris, USA). Single-strand overhangs were removed, DNA fragments were repaired for any damage, and their ends were polished. They were then ligated with stem-loop adapters suitable for PacBio sequencing. Fragments that failed to link were removed using an exonuclease, and target fragments were screened using the BluePippin system (Sage Science, USA). The SMRTbell library was subsequently purified using AMPure PB, and the size of library fragments was assessed using the Agilent 2100 Bioanalyzer (Agilent Technologies, USA). Sequencing was carried out using a PacBio Sequel II instrument with the Sequel II Sequencing Kit 2.0 on Nextomics.