PacBio sequencing is a third generation sequencing technology under active development by Pacific Biosciences. This technology uses zero-mode waveguides - a small well with a DNA polymerase immobilised in its base. This allows the incorporation of fluorescently-labelled nucleotides. The fluorescence can be identified by illuminating the zero-mode waveguide - due to its diameter being similar to the wavelength of light, the fluorescence is magnified, removing the need for cluster amplification (as is used in Illumina sequencing). This removes the need for a library preparation process.
SMRT-bell adapters are used to circularise dsDNA molecules, producing long, linear transcripts with the DNA polymerase. This allows the assembly of consensus sequences, helping to remove errors (as PacBio sequencing has a slightly higher error rate compared to Illumina sequencing). By using these adaptors, the dsDNA can be displaced by the DNA polymerase, producing circular molecules that can be replicated many times.
Although SMRT-bell adapters may be required, the library preparation steps are much less time consuming than Illumina sequencing. Illumina sequencing requires the addition of adaptor molecules to short sections of DNA (approximately 400bp), hybridisation to a flow cell, and cluster amplification (similar to PCR resulting in clusters of identical DNA strands after restriction). This process takes time, and requires the purchase of single-use adaptor molecules and flow cells from Illumina. PacBio sequencing requires only the zero-mode waveguides, SMRT-bell adapters, and fluorescent nucleotides. This allows for a simpler preparation process, as well as producing consensus sequences (where SMRT-bell adapters are used), while taking less time.
PacBio has considerably longer read lengths than Illumina sequencing. Although PacBio is a sequencing-by-synthesis process, it does not involve terminator nucleotides, allowing for continuous reads. This allows for considerably longer read lengths, up to 50 kb (compared to ~150 bp with Illumina sequencing). In genomes with lengthy repeat regions, including the human genome, this allows larger sections of the genome to be resolved, reducing the possibility of obtaining a discontinuous sequence. PacBio sequencing was used during the telomere-to-telomere project, due to the ability to resolve longer repeat regions. A limitation of PacBio sequencing would be genomes with incredibly long repeat regions - including telomeres and centromeres. This requires longer read lengths which may be offered by other third generation sequencing technologies, such as Oxford Nanopore.
PacBio sequencing is a real-time sequencing technology, removing the requirement to computationally assemble the sequence. This reduces the time taken to sequence a genome.