Single Molecule, Real-Time (SMRT) sequencing technology
PacBio’s Single Molecule, Real-Time (SMRT) sequencing technology uses nanowells called zero-mode waveguides (ZMWs) to record the incorporation of fluorescently-labeled nucleotides onto single DNA molecules in real-time. The polymerase-bound template is immobilized at the bottom of a ZMW and as the polymerase incorporates nucleotides, light is emitted and detected, allowing real-time monitoring of nucleotide incorporation. Each Revio SMRT cell houses 25 million ZMWs, each able to hold a molecule of library DNA.
To achieve high-fidelity (HiFi) reads, the Circular Consensus Sequencing (CCS) approach circularizes DNA fragments using hairpin adapters, allowing for multiple, continuous passes of the DNA polymerase over the template. This repeated sequencing generates a consensus sequence from the multiple reads within a single ZMW, ultimately resulting in highly accurate, long-read data.
Revio
The PacBio Revio is a high-throughput, DNA sequencing platform that delivers long and highly accurate “HiFi” reads with >99.9% accuracy (Q30+) and read lengths commonly exceeding 10 kb. HiFi sequencing is ideal for resolving large and complex genomes and enables comprehensive genomic analysis, including de novo assembly, structural variant discovery, isoform/transcriptome profiling, and epigenetic analysis. Revio produces uniform coverage with minimal GC bias and significantly increases data output and throughput compared to previous PacBio sequencers.
The PacBio genomic DNA library preparation process does not utilize amplification techniques and the resulting library molecules are directly used as templates for the sequencing process. As such, the quality of the starting material will be directly reflected in the extent of sequencing success or failure. Any unrepaired or irreversible DNA damage present in the input material (e.g., inter-strand crosslinks, nicks, etc.) will result in impaired performance. High-quality (i.e., high-molecular-weight genomic as well as high-purity) DNA is imperative for obtaining long read lengths and optimal sequencing performance. PacBio therefore recommends sample preparation using their Nanobind DNA extraction kits.
Additional Resources:
Infographic: SMRT sequencing — How it works