SMRT-Sequencing
The principle of PacBio SMRT sequencing:
The PacBio sequencing uses the SMRT (Single molecule Real-Time Sequencing Technology) to sequence log reads with uniform coverage. The SMRT technology uses the zero-mode waveguide (ZMW) nanostructure to sequence the DNA.
Precisely, within ZMW chamber, a molecule of DNA polymerase and DNA template to be sequenced is adsorbed to the bottom of the base, and nucleotides tagged with fluorescent molecules are released. The light is shined from the below ZMW chamber. Only the very bottom of the detection chamber is reached by the decaying light, meaning only the DNA polymerase molecule and whatever base that is currently attached to it can be illuminated and detected. This video gives an overview of the technology.
Library preparation
* Please contact Vanessa Schmid at the sequencing core for pricing information
Application areas of SMRT sequencing
- Whole genome sequencing
- RNA sequencing
- Targeted Sequencing
- Epigenetics
- Metagenomics
How the sequencing works
(source :Eid J., Fehr A., Gray J., Luong K., Lyle J., Otto G., Peluso P., Rank D., Baybayan P., Bettman B., et al.Real-time DNA sequencing from single polymerase molecules, Science, 2009, vol. 323 (pg. 133-138))
The SMRTbell (grey) as produced in the last step of library preparation is diffused into a ZMW well and adapter binds to a polymerase immobilized at the bottom of the well. Million such wells are present in the sequel system. To maximize the throughput, each well must receive one such SMRTbell.
In the above figure, four types of nucleotides are labeled with different fluorescent dyes. 1) a nucleotide forms an association with the template strand at the polymerase active site. 2) this causes fluorescence at the site which is captured in color channel 3) after the formation of phosphodiester bond, the dye diffuses out of the well thus ending fluorescence 4) polymerase then translocates to the next position 5) next nucleotide forms an association at the polymerase active site.
Bioinformatics analysis
SMRT sequencing enables various types of bioinformatics analysis such as:
- De Novo Assembly
- Transcriptome analysis
- Base Modification analysis
- Minor variants and phasing analysis
For bioinformatics analysis, we highly recommend that you come by our lab or contact us so that we can discuss the details of your experiment.
Features of the PacBio Sequel Systems
| Supported SMRT Cell | SMRT Cell 1M |
|---|---|
| Number of HiFi Reads >99%* Accuracy | Up to 500,000 |
| Sequencing Run Time per SMRT Cell | Up to 20 hrs |
| Recommended Chemistry | 3 |
| Instrument Control Software | v8.0 |
| SMRT Link | v8.0 |
Advantages of SMRT Sequencing
- Single molecule resolution
- Long read
- Speed
- High accuracy
This paper provides an overview of the advantages of SMRT sequencing
References
- Sedlazeck, Fritz J et al. (2018) Accurate detection of complex structural variations using single-molecule sequencing. Nature methods
- Chaisson, Mark J P et al. (2015) Genetic variation and the de novo assembly of human genomes. Nature reviews. Genetics
- Rhoads, Anthony et al. (2015) PacBio sequencing and its applications. Genomics, proteomics & bioinformatics
- Eid J., Fehr A., Gray J., Luong K., Lyle J., Otto G., Peluso P., Rank D., Baybayan P., Bettman B., et al.Real-time DNA sequencing from single polymerase molecules, Science, 2009, vol. 323 (pg. 133-138)