title: Single-molecule sequencing: Apocynum venetum leaves
design: The extraction of DNA was conducted using the CTAB method. The quality of the extracted genomic DNA was checked by 1% agarose gel electrophoresis, and the concentration was quantified using a Qubit fluorimeter.  Long-read sequencing was performed with a PacBio Sequel sequencer (Pacific Biosciences, Menlo Park, CA, USA). The SMRT Bell library was prepared using a DNA Template Prep Kit 1.0 and two 20 kb SMRTbell libraries were constructed. Genomic DNA (10 g) was mechanically sheared using a Covaris g-Tube with a goal of DNA fragments of approximately 20 kb. A Bioanalyzer 2100 12K DNA Chip assay was used to assess the fragment size distribution. Sheared genomic DNA (5 g) was DNA-damage repaired and end-repaired using polishing enzymes. A blunt-end ligation reaction followed by exonuclease treatment was conducted to generate the SMRT Bell template. A Blue Pippin device (Sage Science, Inc., Beverly, MA, USA) was used to size select the SMRT Bell template and enrich large fragments (> 10 kb). The size-selected library was quality inspected and quantified on an Agilent Bioanalyzer 12 kb DNA Chip  and a Qubit fluorimeter . A ready-to-sequence SMRT Bell-Polymerase Complex was created using a Binding Kit 2.0, according to the manufacturers instructions. The Sequel instrument was programmed to load and sequence the sample on PacBio SMRT cells v3.0 , acquiring one movie of 360 min per SMRT cell. The MagBead loading method was employed to improve the enrichment of the larger fragments.