2.2. Library construction and sequencing

Purified genomic DNA (gDNA) was used for library construction with the SMRTbell Express Template Prep Kit (Pacific Biosciences, Cat. No. 101-357-000). In brief, gDNA was mechanically sheared to an average size of 20 kb using a Covaris g-TUBE device (Part No. 520079). In total, 5 μg of sheared gDNA was damage-repaired and end-repaired using polishing enzymes. Blunt-end adapter ligation was used to create the SMRTbell template. Adapter dimers and contaminants were removed using the AMPure XP bead purification system (Beckman Coulter, Cat. No. A63882). A BluePippin size selection system (Sage Science, Cat. No. BLU0001) was used to size select the SMRTbell template and enrich for fragments > 15 kb. Sequencing primer v4 was annealed to the SMRTbell template, and a DNA polymerase/template complex was created using the Sequel Binding Kit 2.1 (Pacific Biosciences, Cat. No. 101-365-900). An additional AMPure XP purification step was performed to remove excess primer and polymerase prior to sequencing. The library was sequenced on a Sequel instrument using SMRT Cell 1M v2 (Pacific Biosciences), taking one movie of 10 hours per cell with the Sequel Sequencing Kit 2.0 (Pacific Biosciences).