PACBIO sequence P. oligandrum genome
Purification steps were performed using 0.45X AMPure PB beads (PacBio). A total of 10 g of DNA was purified then sheared at 40 kb using the megaruptor system (Diagenode). Using SMRTBell template Prep Kit 1.0 (PacBio), a DNA and END damage repair step was performed on 5 g of sample. Then blunt hairpin adapters were ligated to the library. The library was treated with an exonuclease cocktail to digest unligated DNA fragments. A size selection step using a 10 kb cutoff was performed on the BluePippin Size Selection system (Sage Science) with the 0.75 % agarose cassettes, Marker S1 high Pass 15-20 kb. Conditioned Sequencing Primer V2 was annealed to the size-selected SMRTbell. The annealed library was then bound to the P6-C4 polymerase using a ratio of polymerase to SMRTbell at 10:1. Then after a magnetic bead-loading step (OCPW), SMRTbell libraries were sequenced on 6 SMRTcells on RSII instrument at 0.18 to 0.23 nM with a 360 min movie. Sequencing summary for each SMRTcells are presented in Table 1.