PacBio sequencing of Heterodera humuli: eggs and J2s
High molecular DNA from 10,200 nematodes (both eggs and J2) was extracted using the MagAttract HMW DNA (Qiagen, Hilden, Germany) kit. Purified genomic DNA sheared with Megaruptor 3 (Diagenode, Denville, NJ, USA). Then, it was amplified using the PacBio Ultra-Low DNA input kit following manufacturers protocol (Pacific Biosciences, Menlo Park, CA, USA) and converted into a PacBio library with the SMRTbell Express Template Prep kit version 2.0 (PacBio Biosciences, Menlo Park, CA, USA). Library quantification was performed with Qubit and fragment size distribution was analyzed on a Femto Pulse. Sequencing was carried out on one SMRT cell 8 M on a PacBio Sequel IIe system using the circular consensus sequencing (CCS) mode and a 30-h movie time with default parameters, which include three minimum passes and a minimum accuracy of 99%.