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Introduction to Pacific Biosciences (PACBIO) Single Molecule Real Time (SMRT) Sequencing Technology

Pacific Biosciences (PacBio) single molecule real time (SMRT) sequencing technology, also termed as third generation sequencing technology, enables the sequencing of very long reads. The defining feature of this technology is the capacity to sequence long reads in real time during DNA polymerisation, with no requirement for amplification prior sequencing.
The costs for hardware, consumables and cost per sequenced nucleotide, however, are slightly higher compared to most reasonable second generation sequencing technologies.

How does Pacific Biosciences Single Molecule Real Time (SMRT) sequencing technology works?

The Single Molecule Real Time (SMRT) sequencing technology is based on incorporation of phospholinked fluorescent labelled nucleotides into a growing DNA strand. This process takes place in a nanophotonic visualization chambers (well) and is performed by an immobilized polymerase enzyme during the DNA replication step. Each type of nucleotides (A, G, T, C) is labelled with different fluorescent linkers resulting in different light signals during nucleotide incorporation. During incorporation of analogue nucleotide into the DNA strand a specific light signal is induced by the fluorescent label before it is cleaved away by the polymerase. The Zero Mode Waveguide (ZMW) allows only to illuminate light on the bottom of each well. In each well a single DNA polymerase/template complex is immobilized. The light signals that arises during incorporation of the different nucleotides will be monitored and translated into sequence information. Thousands of different DNA polymerase/template complexes can be analyzed simultaneously in one SMRT cell.
The following video demonstrates how SMRT technology works.

Pacific Biosciences sequencing time, read length and throughput

The sequencing run time for one SMRT cell is between 30 minutes and 6 hours. The rapid sequencing step enables complete sequencing from sample to result within 24-48 hours.
The average read length is between 10 and 20 kb with a maximum read length of about 60 kb.
Each individual SMRT cell will generate about 55.000 reads with a maximum data output between 500 Mb and 1Gb. The PacBio RS II and the Sequel™ system both allows to sequence up to 16 SMRT cells in one run. The total read number per RS II run can be up to 800.000 reads. Even though the total read number per run are markedly lower compared with other technologies, calculating an average read length of 20.000 nucleotides would corresponds to 80 million 200 nucleotide reads as provides by most second generation sequencing platforms.
The Sequel™ System enables about 7x more reads with 1 million zero-mode waveguides (ZMWs) per SMRT cell.

Pacific Biosciences sequencing platforms and its applicationsPACBIORSII

Pacific Biosciences offers two different sequencing systems the PacBio RS II and the lately (September 2015) launched Sequel™ System. The Sequel™ System provides higher throughput, more scalability, a reduced footprint and lower sequencing project costs compared with the PACBIO RS II platform.
A major application of this technology is whole genome sequencing of small genomes such as bacterial or viral genomes in order to identify haplotypes or perform quasi species analysis. This technology can be implemented for example for investigation in RNA viruses with high quasi species (e.g., HCV and HIV).
Further application fields are targeted sequencing, complex population analysis, RNA sequencing of targeted transcripts, and microbial epigenetics. Utilization of this technology will be especially beneficial for sequencing of complex regions containing structural variants such as insertions, deletions, duplications, recombination, inversions or repetitive elements.
For more detailed information on PacBio sequencing systems and its applications visit the Pacific Biosciences website.