This is a companion article to the feature, “Assay Development for Clinical Genomics.”
Figure 1. A step-wise illustration of single-molecule, real-time (SMRT) sequencing: (1) fluorescent phospholinked labeled nucleotides are introduced into the zero-mode waveguide; (2) the base being incorporated is held in the detection volume for tens of milliseconds, producing a bright flash of light; (3) the phosphate chain is cleaved, releasing the attached dye molecule; (4–5) the process repeats. Click to expand.
Single-molecule, real-time (SMRT) sequencing is a technology from Pacific Biosciences, Menlo Park, Calif, that produces extremely long reads. The mean read length is about 10 kb, with many reads stretching to 50 kb or longer.
The sequencer works by affixing a DNA polymerase to the bottom of a zero-mode waveguide, a structure designed to allow observation of a single base as it is being incorporated by the polymerase (see Figure 1). Each of the four DNA bases is assigned a different fluorescent dye, so as each new base is incorporated, it gives off a distinct, identifiable signal. After incorporation, the fluorescent tag is removed and the next base is added.
Figure 2. DNA polymerase in the zero-mode waveguide as SMRT sequencing occurs.
Sequencers utilizing this technology include the PacBio RS II and Sequel systems, which use SMRT cells that contain as many as 1 million zero-mode waveguides (see Figure 2).
After sequencing, analysis tools assemble the data using the longest reads as anchors, with shorter reads layered on top to achieve very high consensus accuracy. This approach has generated some of the longest contiguous genome assemblies ever produced, including a diploid human genome assembly produced by scientists at Mount Sinai and collaborators at other institutions that was published in 2015.
