Pacific Biosciences

Single Molecular Real-Time (SMRT) sequencing employs a specialized flow cell with many thousands of individual picolitre wells with transparent bottoms -- zero-mode waveguides (ZMW). The polymerase is fixed to the bottom of the well and allows the DNA strand to progress through the ZMW. As a result, the system can focus on a single molecular. SMRT sequencing allows for real-time imaging of fluorescently tagged nucleotides that are synthesized along individual DNA template molecules. The sequencing reaction ends when the template and polymerase dissociate. The average read length from the PacBio instrument is approximately 2 kb, and some reads may be over 20 kb. Longer reads are especially useful for de novo assemblies of novel genomes that can span many more repeats and bases.

Highly repetitive elements found in both eukaryotic and prokaryotic genomes pose a challenge for genome assembly and make the detailed study of repetitive sequences difficult. Long-read sequencing delivers reads in excess of several or dozens of kilobases (kbs), which can span complex or repetitive regions with a single continuous read, allowing for the resolution of these large structural features. Besides considerably longer and highly accurate DNA sequences from individual unamplified molecules, it can also exhibit where methylated bases occur, thereby providing functional information about DNA methyltransferases encoded by the genome.