Genomic DNA was sheared to 8 kb using an ultrasonicator (Covaris Inc, Woburn, MA) and was converted into the proprietary SMRTbell™ library format using RS DNA Template Preparation Kit (Pacific Biosciences, Melon Park, CA). Briefly, sheared DNA was end repaired, and hairpin adapters were ligated using T4 DNA ligase. Incompletely formed SMRTbell templates were degraded with a combination of Exonuclease III and Exonuclease VII. The resulting DNA templates were purified using SPRI magnetic beads (AMPure, Agencourt Bioscience, Beverly, MA) and annealed to a two-fold molar excess of a sequencing primer that specifically bound to the single-stranded loop region of the hairpin adapters.
SMRTbell templates were subjected to standard SMRT sequencing using an engineered phi29 DNA polymerase on the PacBio RS system according to manufacturer's protocol. The PacBio RS system continuously monitors zero-mode waveguides (ZMWs) in sets of 75000 at a time. Within each ZMW a single DNA polymerase molecule is attached to the bottom surface such that it permanently resides within the detection volume where it can be watched as it performs sequencing by synthesis. Within each chamber, Phospholinked nucleotides, each type labeled with a different colored fluorophore, are then introduced into the reaction solution at high concentrations that promote enzyme speed, accuracy, and processivity. Pulse calling, utilized a threshold algorithm on the dye weighted intensities of fluorescence emissions, and read alignments, achieved using a Smith-Waterman algorithm. Reads were filtered after alignment to remove low quality sequences derived from doubly-loaded ZMWs.
SMRTbell templates were subjected to standard SMRT sequencing using an engineered phi29 DNA polymerase on the PacBio RS system according to manufacturer's protocol. The PacBio RS system continuously monitors zero-mode waveguides (ZMWs) in sets of 75000 at a time. Within each ZMW a single DNA polymerase molecule is attached to the bottom surface such that it permanently resides within the detection volume where it can be watched as it performs sequencing by synthesis. Within each chamber, Phospholinked nucleotides, each type labeled with a different colored fluorophore, are then introduced into the reaction solution at high concentrations that promote enzyme speed, accuracy, and processivity. Pulse calling, utilized a threshold algorithm on the dye weighted intensities of fluorescence emissions, and read alignments, achieved using a Smith-Waterman algorithm. Reads were filtered after alignment to remove low quality sequences derived from doubly-loaded ZMWs.
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