PacBio long read sequencing of Heterodera trifolii
DNA fragments (20Kb) by shearing genomic DNA followed by the manufacturer's recommendedprotocol were generated, using the covaris G-tube. Small fragments were removed under the AmpureXPbead purification system. A total of 5 g for each sample was used as input into library preparation. TheSMRTbell library was constructed by usin SMRTbell Express Template Preparation Kit(101-357-000).Small fragments were eliminated for large-insert library using the BluePippin Size selection system.Sequencing primer v4 was annealed to the SMRTbell template, and DNA polymerase was bound to thecomplex (Sequel Binding kit 3.0). The complex was purified using AMPure purification for the removal ofexcess primer and polymerase prior to sequencing. The SMRTbell library was sequenced using SMRT cells(Pacific Biosciences) and Sequel Sequencing Kit v3.0. For the capture of each SMRT Cell 1M v3, 1 x 10hour movies were used under the sequel sequencing platform.