PacBio sequencing of Thalassospira sp. strain KO164
Genomic DNA was isolated from KO164 using MoBio UltraClean Extraction kit and sequencing was performed at Joint Genome Facility (Walnut Creek, CA) using PacBio RS II instrument. 5µg of gDNA was used to generate the library and the DNA was sheared using Covaris g-Tubes to generate sheared fragments of >10kb in length. The sheared DNA fragments were then prepared using Pacific Biosciences SMRTbell template preparation kit, where the fragments were treated with DNA damage repair, had their ends repaired so that they were blunt-ended, and 5’ phosphorylated. Pacific Biosciences hairpin adapters were then ligated to the fragments to create the SMRTbell template for sequencing. The SMRTbell templates were then purified using exonuclease treatments and size-selected using AMPure PB beads. Sequencing primer was then annealed to the SMRTbell templates and Version P6 sequencing polymerase was bound to them. The prepared SMRTbell template library was then sequenced on a Pacific Biosciences RSII sequencer using Version C4 chemistry and 2 hour sequencing movie run times.