SMRTCell4 pool of 20 ruminant Mycoplasmas
Genomic DNA extracted using phenol-chloroform method . DNA of each sample was sheared at 9 kb using the megaruptor system (diagenode) and then barcoded by using the SMRTbell Barcoded Adapter Prep Kit.  SMRTbell library was constructed by pooling an equal amount of  the 20 barcoded samples. A size selection step using a 6kb cutoff was performed on the BluePippin Size Selection system (Sage Science) with the 0.75% agarose cassettes, Marker S1 high Pass 6 to 10 kb V3. The sized pool was purified again with AMPure PB beads at 0.45 and quantified. Conditioned Sequencing Primer V2 was annealed to the size-selected SMRTCell library. The annealed library was then loaded on four SMRTCells by using P6C4 chemistry on the PacBio RSII sequencer using 360minute movies.