Pacbio SEQ2 Short insert library: Mixed E. coli, H. pylori and S. cerevisiae, D3
1ug E. coli K12 MG1655 gDNA and 1ug H. pylori J99 gDNA were mixed. Then the mixture was diluted with S. cerevisiae JEL1 gDNA with the ratio of 1:30.55 (D3). The short-insert DNA SMRTbell library was performed according to the manufacturers instructions by first shearing the sample to ~200bp using a Covaris LE220 Focused-ultrasonicator,followed by damage and end repair and ligation of single-stranded barcoded adaptors. Sequencing primer was then annealed to the SMRTbells and incubated with the template for 1 hour at 20 C using a ratio of 20:1. The Sequel 2.1 polymerase was then bound to the annealed template complex using a ratio of 30:1 polymerase to SMRTbell at 0.5 nM for 4 h at 30 C. The binding complex was cleaned up, diluted to the final loading concentration, and held at 4 C until diffusion-based loading on the Sequel II instrument. Diluted binding complex at concentration of 125-175pM was loaded on the 8M sequencing chip and 30 hour movies were recorded according to the manufacturers guidelines.