title: PacBio Hifi-reads of Atelopus laetissimus (Cell 5)
design: The SMRTbell library for HiFi sequencing was prepared at BYU following the instructions provided with the SMRTbell Express Template Prep Kit 2.0. High-molecular-weight (HMW) genomic DNA was fragmented to achieve a desired size range of 15 to 18 kb. The fragmented DNA underwent concentration using 0.45 AMPure PB beads at 37C for 15 minutes to remove single-strand overhangs. Subsequently, the DNA underwent a set of enzymatic procedures. These steps encompass DNA damage repair at 37C for 30 minutes, followed by end repair and A-tailing at 20C for 10 minutes and 65C for 30 minutes. Ligation of Overhang Adapter v3 was carried out at 20C for 60 minutes and 65C for 10 minutes to inactivate the ligase. Lastly, the DNA underwent nuclease treatment at 37C for 1 hour. The resulting SMRTbell library was purified and concentrated using 0.45 AMPure PB beads. Size selection was performed using the BluePippin system to collect fragments larger than 10 kb. The final HiFi SMRTbell library, with an average fragment size of 16 kb, was sequenced using six 8M SMRT cells, Sequel II sequencing Chemistry 2.0, and 30-hour movies for each cell on a PacBio Sequel II sequencer.