Sample and Library Preparation for PacBio Long-Read Sequencing in Grapevine

PacBio long-read sequencing is a third-generation technology that generates long reads up to 20 kilobases (kb), unlike short-read sequencing instruments that produce up to 600 bases. Long-read sequencing is particularly advantageous in higher organisms, such as humans and plants, where repetitive regions in the genome are more abundant. The PacBio long-read sequencing uses a single molecule, real-time approach where the SMRT cells contain several zero-mode waveguides (ZMWs). Each ZMW contains a single DNA molecule bound by a DNA polymerase. All ZMWs are flushed with deoxy nucleotides with a fluorophore specific to each nucleotide. As the sequencing proceeds, the detector detects the wavelength of the fluorescence and the nucleotides are read in real-time. This chapter describes the sample and library preparation for PacBio long-read sequencing for grapevine.