The principle of PacBio SMRT sequencing:
The PacBio sequencing uses the SMRT (Single moelcule 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 of 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.
Application areas of SMRT sequencing
- Whole geneome sequencing
- RNA sequencing
- Targeted Sequencing
How the sequencing works
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 bollom of well. Million such wells are present in the sequel system. To maximize the through-put, each well must receive one such SMRTbell.
In the above figure four types of nucleotide are labelled with different fluorescent dye. 1) a nucleotide forms an association with 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.
SMRT sequenincg 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|
|Instrument Control Software||v8.0|
Advantages of SMRT Sequencing
- Single molecule resolution
- Long read
- High accuracy
This paper provides an overview of advantages of SMRT sequencing
- 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