Analysis

SMRT-Sequencing

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.

Library preparation

smrt-seq
                                                  Library Prepration workflow (https://www.pacb.com/)
* Please contact Vanessa.Schmid@UTSouthwestern.edu at the sequencing core for pricing information

 Application areas of SMRT sequencing

  • Whole geneome sequencing
  • RNA sequencing
  • Targeted Sequencing
  • Epigenetics
  • Metagenomics

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.

Bioinformatics analysis

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
Recommended Chemistry 3
Instrument Control Software  v8.0
SMRT Link v8.0
(source : https://www.pacb.com/products-and-services/sequel-system/ )

Advantages of SMRT Sequencing

  • Single molecule resolution
  • Long read
  • Speed
  • High accuracy

This paper provides an overview of advantages of SMRT sequencing

References