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SMRT sequencing

Using SMRT (Single Molecule, Real-Time) technology, a DNA molecule is attached to the sequencing slide, more specifically at the bottom of nanosize zero-mode waveguide (ZMW)  wells, together with a DNA polymerase. As fluorescent nucleotides are provided to the sequencing slide, the DNA polymerase synthesises a new strand, generating a nucleotide-specific signal for each well. As a result of  the ZMW technology, which in simple terms acts as a ‘pipe’ directly the light signal from the well to the detector, the fluorescence is detected without the need for fragment amplification (essential for SGS which is also based on fluorescence reading). Figure 11 provides a schematic representation of the different steps in SMRT sequencing.

The image breaks down the different steps involved in SMRT sequencing. First, the DNA molecule to sequence is bound by the adapters, creating a circular DNA fragment. The fragment is then placed in the nanowell, where a DNA polymerase synthesise the complementary sequence by incorporating one fluorescent nucleotide at a time. This allows for the sequence to be reconstructed.
Figure 11 Representation of the different steps involved in SMRT sequencing. The fragment to sequence is bound by the adapters, creating a circular DNA fragment. In the nanowell, this is bound by the DNA polymerase, which incorporates the fluorescent nucleotides one at a time, allowing for the sequence to be reconstructed. Image credit: Laura Olivares Boldú, Wellcome Connecting Science. For more information visit this page.


Thanks to SMRT, HiFi PacBio sequencing was the first TGS to reach accuracies greater than 99.5 %, by also carrying the advantage of generating kilobases long reads.