The PacBio libraries were constructed using approximately 10 µg of genomic V. dahliae DNA that was mechanically sheared to a size of ~22 kb, using g-TUBES (Covaris, Inc., Woburn, MA) according the manufacturer’s instructions. PacBio SMRTbell libraries were prepared by ligation of hairpin adaptors at both ends of the DNA fragment (52 (link)) using the PacBio DNA template preparation kit 2.0 (Pacific Biosciences of California, Inc., Menlo Park, CA) for SMRT sequencing on the PacBio RS II machine (Pacific Biosciences of California, Inc.) according to the manufacturer’s instructions. Libraries were purified using Agencourt AMPure beads (Beckman Coulter, Inc., Brea, CA) to remove short inserts of <1.5 kb. Libraries were size selected using the BluePippin preparation system (Sage Science, Beverly, MA) with a minimum cutoff of 7 kb. The sheared DNA and final library were characterized for size distribution using an Agilent Bioanalyzer 2100 (Agilent Technology, Inc., Santa Clara, CA) along with a DNA12000 chip (Agilent Technology, Inc.).
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PacBio Sequencing of Verticillium dahliae
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Other organizations : Wageningen University & Research, KeyGene (Netherlands)
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Variable analysis
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- Mechanical shearing of genomic V. dahliae DNA to a size of ~22 kb using g-TUBES
- Ligation of hairpin adaptors at both ends of the DNA fragment using the PacBio DNA template preparation kit 2.0
- Size selection of the libraries using the BluePippin preparation system with a minimum cutoff of 7 kb
- Size distribution of the sheared DNA and final library, characterized using an Agilent Bioanalyzer 2100 with a DNA12000 chip
- Genomic V. dahliae DNA amount (approximately 10 µg)
- Manufacturer's instructions for using g-TUBES, PacBio DNA template preparation kit 2.0, and BluePippin preparation system
- No positive or negative controls were explicitly mentioned.
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