Long Read Sequencing (PacBio)

Whole genome sequencing was performed on a Pacific Biosciences RSII platform. The sequencing library was prepared using the SMRTbell^TM Template Prep Kit (Pacific Biosciences, Menlo Park, CA, United States) following manufacturer’s protocol. 5 μg of DNA was fragmented using gTUBE (Covaris Inc., Woburn, MA, United States) to ∼20 kb. After DNA damage repair and ends repair, blunt hairpin adapters were ligated to the template. Non-ligated products were digested by ExoIII and ExoVII exonucleases. Resulting SMRTbell template were purified with AMPure PB beads and size selected on BluePippin system (Sage Science, Beverly, MA, United States), using 0.75% dye-free agarose cassette, with 4–10 kb Hi-Pass protocol and lower cut set on 4 kb. Size selected purified libraries were quantified by Qubit dsDNA High Sensitivity assay. After primer annealing, and P6 polymerase binding, templates were bound to MagBeads for loading. Each sample was sequenced on two SMRT cells, using C4 sequencing kit and 360-min movies per SMRT cell. Presence of unidentified contaminant in two libraries (NSV2 16 days) inhibited sequencing reactions, which manifested in extremely low P1 (2%) and super short reads. Both libraries were subjected to the cleanup procedure that involves binding annealed SMRTbell libraries to magnetic beads, washing the bound annealed DNA SMRTbell templates to remove potential contaminants, and eluting the purified, annealed DNA SMRTbell templates from the magnetic beads. The purified SMRTbell templates were then re-quantified by Qubit and prepared for sequencing on the PacBio RSII according to the Binding Calculator. After cleanup procedure, PacBio RS II instrument sequencing yields were comparable to the other samples. Raw sequencing data are available on request.