title: Whole Genome Shotgun Sequencing of Switchgrass (Panicum virgatum AP13)
design: Switchgrass (Panicum virgatum AP13 (tetraploid)) DNA was isolated using a modified protocol for Chen and Ronald [1999]. Library preparation and sequencing was performed according to manufacturer’s instructions (Pacific Biosciences, Menlo Park, CA). In short, 3-10 µg of genomic DNA was isolated and fragmented to 7-10 kbp fragments using HydroShear for 15 minutes. The first of five Ampure XP bead purifications was performed (0.45X Ampure beads added to DNA dissolved in 200 µL EB, vortexed for 10 minutes at 2,000 rpm, followed by two washes with 70% alcohol and finally diluted in EB) After each Ampure XP purification step a quality control was performed comprised of DNA concentration determination by nanodrop and fragment size distribution by bioanalyzer. Next, the DNA fragments were repaired using DNA Damage Repair solution (1X DNA Damage Repeat Buffer, 1X NAD+, 1 mM ATP high, 0.1 mM dNTP, and 1X DNA Damage Repeat Mix with a final volume of 85.3 µL) with a volume of 21.1 µL and incubated at 37oC for 20 minutes. DNA ends were repaired next by adding 1X End Repeat Mix to the solution, which was incubated at 25oC for 5 minutes, followed by the second Ampure XP purification step. Next, 0.75 µM of Blunt Adapter was added to the DNA, followed by 1X template Prep Buffer, 0.05 mM ATP low and 0.75 U/µL T4 ligase to ligate (final volume of 47.5 µL) the bell adapters to the DNA fragments. This solution was incubated at 25oC for 30 minutes, followed by a 65oC 10 minute heat-shock. The exonuclease treatment to remove unligated DNA fragments consists of 1.81 U/µL Exo III and 0.18 U/µL Exo IV (final volume of 3.8 µL) which is incubated at 37oC for 1 hour. Next, three Ampure XP purifications steps were performed. Finally, the bell primer is annealed to the PacBio bell with inserted DNA fragment (80oC for 2 minute 30 followed by decreasing the temperature by 0.1o/s to 25Co). This complex was loaded into PacBio RS SMRT cells, which were loaded onto the machine for 75 minute recording.